Therapeutic uses of pharmaceutical compositions comprising cyclic boronic acid ester derivatives

ABSTRACT

Disclosed herein are antimicrobial compounds compositions, pharmaceutical compositions, the use and preparation thereof. Some embodiments relate to cyclic boronic acid ester derivatives and their use as therapeutic agents.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/935,782filed Mar. 26, 2018 which is a division of U.S. application Ser. No.15/624,473 filed Jun. 15, 2017 now U.S. Pat. No. 10,004,758 issued Jun.26, 2018 which is a continuation of U.S. application Ser. No. 15/015,002filed Feb. 3, 2016 now U.S. Pat. No. 9,694,025 issued Jul. 4, 2017 whichis a division of U.S. application Ser. No. 13/953,647 filed Jul. 29,2013 now U.S. Pat. No. 9,296,763 which is a division of U.S. applicationSer. No. 13/205,112 filed Aug. 8, 2011 entitled “CYCLIC BORONIC ACIDESTER DERIVATIVES AND THERAPEUTIC USES THEREOF” now U.S. Pat. No.8,680,136 which claims the benefit of U.S. Prov. App. Nos. 61/372,296,filed Aug. 10, 2010, and 61/488,655, filed May 20, 2011, all of theforegoing are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to antimicrobial compounds, compositions,their use and preparation as therapeutic agents. In particular, thepresent invention relates to cyclic boronic acid ester compounds.

BACKGROUND OF THE INVENTION

Antibiotics have been effective tools in the treatment of infectiousdiseases during the last half-century. From the development ofantibiotic therapy to the late 1980s there was almost complete controlover bacterial infections in developed countries. However, in responseto the pressure of antibiotic usage, multiple resistance mechanisms havebecome widespread and are threatening the clinical utility ofanti-bacterial therapy. The increase in antibiotic resistant strains hasbeen particularly common in major hospitals and care centers. Theconsequences of the increase in resistant strains include highermorbidity and mortality, longer patient hospitalization, and an increasein treatment costs.

Various bacteria have evolved β-lactam deactivating enzymes, namely,β-lactamases, that counter the efficacy of the various β-lactams.β-lactamases can be grouped into 4 classes based on their amino acidsequences, namely, Ambler classes A, B, C, and D. Enzymes in classes A,C, and D include active-site serine β-lactamases, and class B enzymes,which are encountered less frequently, are Zn-dependent. These enzymescatalyze the chemical degradation of β-lactam antibiotics, renderingthem inactive. Some β-lactamases can be transferred within and betweenvarious bacterial strains and species. The rapid spread of bacterialresistance and the evolution of multi-resistant strains severely limitsβ-lactam treatment options available.

The increase of class D β-lactamase-expressing bacterium strains such asAcinetobacter baumannii has become an emerging multidrug-resistantthreat. A. baumannii strains express A, C, and D class β-lactamases. Theclass D β-lactamases such as the OXA families are particularly effectiveat destroying carbapenem type β-lactam antibiotics, e.g., imipenem, theactive carbapenems component of Merck's Primaxin® (Montefour, K.; et al.Crit. Care Nurse 2008, 28, 15; Perez, F. et al. Expert Rev. Anti Infect.Ther. 2008, 6, 269; Bou, G.; Martinez-Beltran, J. Antimicrob. AgentsChemother. 2000, 40, 428. 2006, 50, 2280; Bou, G. et al, J. Antimicrob.Agents Chemother. 2000, 44, 1556). This has imposed a pressing threat tothe effective use of drugs in that category to treat and preventbacterial infections. Indeed the number of catalogued serine-basedβ-lactamases has exploded from less than ten in the 1970s to over 300variants. These issues fostered the development of five “generations” ofcephalosporins. When initially released into clinical practice,extended-spectrum cephalosporins resisted hydrolysis by the prevalentclass A β-lactamases, TEM-1 and SHV-1. However, the development ofresistant strains by the evolution of single amino acid substitutions inTEM-1 and SHV-1 resulted in the emergence of the extended-spectrumβ-lactamase (ESBL) phenotype.

New β-lactamases have recently evolved that hydrolyze the carbapenemclass of antimicrobials, including imipenem, biapenem, doripenem,meropenem, and ertapenem, as well as other β-lactam antibiotics. Thesecarbapenemases belong to molecular classes A, B, and D. Class Acarbapenemases of the KPC-type predominantly in Klebsiella pneumoniaebut now also reported in other Enterobacteriaceae, Pseudomonasaeruginosa and Acinetobacter baumannii. The KPC carbapenemase was firstdescribed in 1996 in North Carolina, but since then has disseminatedwidely in the US. It has been particularly problematic in the New YorkCity area, where several reports of spread within major hospitals andpatient morbidity have been reported. These enzymes have also beenrecently reported in France, Greece, Sweden, United Kingdom, and anoutbreak in Germany has recently been reported. Treatment of resistantstrains with carbapenems can be associated with poor outcomes.

Another mechanism of β-lactamase mediated resistance to carbapenemsinvolves combination of permeability or efflux mechanisms combined withhyper production of beta-lactamases. One example is the loss of a porincombined in hyperproduction of ampC beta-lactamase results in resistanceto imipenem in Pseudomonas aeruginosa. Efflux pump over expressioncombined with hyperproduction of the ampC β-lactamase can also result inresistance to a carbapenem such as meropenem.

Because there are three major molecular classes of serine-basedβ-lactamases, and each of these classes contains significant numbers ofβ-lactamase variants, inhibition of one or a small number ofβ-lactamases is unlikely to be of therapeutic value. Legacy β-lactamaseinhibitors are largely ineffective against at least Class Acarbapenemases, against the chromosomal and plasmid-mediated Class Ccephalosporinases and against many of the Class D oxacillinases.Therefore, there is a need for improved β-lactamase inhibitors.

SUMMARY OF THE INVENTION

The present invention relates to antimicrobial agents and potentiatorsthereof. Some embodiments include compounds, compositions,pharmaceutical compositions, use and preparation thereof. In particular,some embodiments, relate to cyclic boronic acid ester derivatives.

Some embodiments include compounds having the structure of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   Y is a 1-4 atom alkylene or 2-4 atom alkenylene linker,        optionally substituted by one or more substituents selected from        the group consisting of Cl, F, CN, CF₃, —R⁹, —OR⁹, —C(═O)NR⁹R¹⁰,        and —C(═O)OR⁹, wherein said alkylene or alkenylene linker is        optionally fused to an optionally substituted aryl, optionally        substituted heteroaryl, optionally substituted carbocyclyl, or        optionally substituted heterocyclyl;    -   R¹ is selected from a group consisting of —C₁₋₉alkyl,        —C₂₋₉alkenyl, —C₂₋₉alkynyl, —NR⁹R¹⁰, —C₁₋₉alkylR¹¹,        —C₂₋₉alkenylR¹¹, —C₂₋₉alkynylR¹¹, -carbocyclyl-R¹¹,        —CH(OH)C₁₋₉alkylR⁹, —CH(OH)C₂₋₉alkenylR⁹, —CH(OH)C₂₋₉alkynylR⁹,        —CH(OH)carbocyclyl-R⁹, —C(═O)R⁹, —C(═O)C₁₋₉alkylR⁹,        —C(═O)C₂₋₉alkenylR⁹, —C(═O)C₂₋₉alkynylR⁹,        —C(═O)C₂₋₉carbocyclyl-R⁹, —C(═O)NR⁹R¹⁰, —N(R⁹)C(═O)R⁹,        —N(R⁹)C(═O)NR⁹R¹⁰, —N(R⁹)C(═O)OR⁹, —N(R⁹)C(═O)C(═NR¹⁰)R⁹,        —N(R⁹)C(═O)C(═CR⁹R¹⁰)R⁹, —N(R⁹)C(═O)C₁₋₄alkylN(R⁹)C(═O)R⁹,        —N(R⁹)C(═NR¹⁰)R⁹, —C(═NR¹⁰)NR⁹R¹⁰, —N═C(R⁹)NR⁹R¹⁰, —N(R⁹)SO₂R⁹,        —N(R⁹)SO₂NR⁹R¹⁰, —N═CHR⁹, substituted or unsubstituted aryl,        substituted or unsubstituted heteroaryl, substituted or        unsubstituted carbocyclyl, and substituted or unsubstituted        heterocyclyl;    -   R⁶ is selected from a group consisting of H, —C₁₋₉alkyl,        C₂₋₉alkenyl, —C₂₋₉alkynyl, carbocyclyl, —C₁₋₉alkylR¹¹,        —C₂₋₉alkenylR¹¹, —C₂₋₉alkynylR¹¹, carbocyclyl-R¹¹, —C(═O)OR⁹,        —C₁-9alkylCO₂R⁹, —C₂₋₉alkenylCO₂R⁹, —C₂₋₉alkynylCO₂R⁹, and        -carbocyclyl-CO₂R⁹, or alternatively:        -   (i) R⁶ and an R⁷ are taken together with the atoms to which            they are attached to form a substituted or unsubstituted            carbocyclyl or substituted or unsubstituted heterocyclyl,        -   (ii) R⁶ and a carbon atom in Y are taken together with            intervening atoms to form a substituted or unsubstituted            carbocyclyl or substituted or unsubstituted heterocyclyl, or        -   (iii) R⁶ is absent when the carbon to which it is attached            is a ring atom in an aryl or heteroaryl ring;    -   each R⁷ is independently selected from a group consisting of H,        halo, —C₁₋₉alkyl, —C₂₋₉alkenyl, —C₂₋₉alkynyl, —NR⁹R¹⁰, —OR⁹,        —C₁₋₉alkylCO₂R⁹, —C₂₋₉alkenylCO₂R⁹, —C₂₋₉alkynylCO₂R⁹, and        -carbocyclyl-CO₂R⁹, or independently:        -   (i) R⁶ and an R⁷ are taken together with the atoms to which            they are attached to form a substituted or unsubstituted            carbocyclyl or substituted or unsubstituted heterocyclyl,        -   (ii) R⁷ and an R⁸ are taken together with the atoms to which            they are attached to form a substituted or unsubstituted            carbocyclyl or substituted or unsubstituted heterocyclyl,        -   (iii) an R⁷ and a carbon atom in Y are taken together with            intervening atoms to form a substituted or unsubstituted            carbocyclyl or substituted or unsubstituted heterocyclyl,        -   (iv) each of the following conditions are met:            -   (a) Y is a 3-4 atom alkylene or 3-4 atom alkenylene                linker,            -   (b) R⁶ is absent,            -   (c) R⁷ and a carbon atom in Y are taken together with                intervening atoms to form a substituted or unsubstituted                aryl or a substituted or unsubstituted heteroaryl, and            -   (d) each R⁸ attached to a ring atom forming part of the                substituted or unsubstituted aryl or a substituted or                unsubstituted heteroaryl formed by R⁷ and Y is absent;    -   each R⁸ is independently selected from a group consisting of H,        halo, —C₁₋₉alkyl, —C₂₋₉alkenyl, —C₂₋₉alkynyl, —NR⁹R¹⁰, —OR⁹,        —C₁₋₉alkylCO₂R⁹, —C₂₋₉alkenylCO₂R⁹, —C₂₋₉alkynylCO₂R⁹,        -carbocyclyl-CO₂R⁹, or independently:        -   (i) an R⁷ and an R⁸ are taken together with the atoms to            which they are attached to form a substituted or            unsubstituted carbocyclyl or substituted or unsubstituted            heterocyclyl,        -   (ii) a geminal R⁷ and R⁸ together form-C₂₋₉alkenylenylCO₂R⁹,            or        -   (iii) each R⁸ attached to a ring atom forming part of a            substituted or unsubstituted aryl is absent;    -   each R⁹ is independently selected from a group consisting of H,        —C₁₋₉alkyl, C₂₋₉alkenyl, —C₂₋₉alkynyl, carbocyclyl,        —C₁₋₉alkylR¹¹, —C₂₋₉alkenylR¹¹, —C₂₋₉alkynylR¹¹,        -carbocyclyl-R¹¹, substituted or unsubstituted aryl, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        carbocyclyl, and substituted or unsubstituted heterocyclyl;    -   each R¹⁰ is independently selected from a group consisting of H,        —C₁₋₉alkyl, —OR⁹, —CH(═NH), —C(═O)OR⁹, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted carbocyclyl, and substituted or        unsubstituted heterocyclyl;    -   each R¹¹ is independently selected from a group consisting of        substituted or unsubstituted aryl, substituted or unsubstituted        heteroaryl, substituted or unsubstituted carbocyclyl, and        substituted or unsubstituted heterocyclyl; X is selected from a        group consisting of H, —CO₂R¹², and carboxylic acid isosteres;    -   R¹² is selected from a group consisting of H, C₁₋₉alkyl,        —(CH₂)₀₋₃—R₁₁, —C(R¹³)₂OC(O)C₁₋₉alkyl, —C(R¹³)₂OC(O)R¹¹,        —C(R¹³)₂OC(O)OC₁₋₉alkyl and —C(R¹³)₂OC(O)OR¹¹;    -   each R¹³ is independently selected from a group consisting of H        and C₁₋₄alkyl; and    -   m is independently zero or an integer from 1 to 2,    -   wherein each C₁₋₉alkyl, C₂₋₉alkenyl, and C₂₋₉alkynyl is        independently optionally substituted.

In some embodiments, the compound of formula I has the structure offormula II:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   the bond represented by a dashed and solid line represents a        bond selected from the group consisting of a single bond and a        double bond with the proviso that the dashed and solid line can        only be a double bond when n is 1;    -   R² and R⁴ are independently selected from a group consisting of        Cl, F, CN, CF₃, —R⁹, —OR⁹, —C(═O)NR⁹R¹⁰, and —C(═O)OR⁹; or        alternatively, R² and R⁴ are taken together with the atoms to        which they are attached to form a substituted or unsubstituted        aryl, substituted or unsubstituted heteroaryl, substituted or        unsubstituted carbocyclyl or substituted or unsubstituted        heterocyclyl;    -   R³ and R⁵ are independently selected from a group consisting of        Cl, F, CN, CF₃, —R⁹, —OR⁹, —C(═O)NR⁹R¹⁰, and —C(═O)OR⁹, with the        proviso that if the bond represented by a dashed and solid line        is a double bond then R³ and R⁵ are absent; and    -   n is independently zero or an integer from 1 to 2.

In some embodiments, the compound of formula I the structure of formulaIIIa or IIIb:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   the bond represented by a dashed and solid line represents a        bond selected from the group consisting of a single bond and a        double bond;    -   each R² and R⁴ are independently selected from a group        consisting of Cl, F, CN, CF₃, —R⁹, —OR⁹, —C(═O)NR⁹R¹⁰, and        —C(═O)OR⁹; or alternatively, an R² and R⁴ are taken together        with the atoms to which they are attached to form a substituted        or unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted carbocyclyl or substituted or        unsubstituted heterocyclyl;    -   each R³ and R⁵ are independently selected from a group        consisting of Cl, F, CN, CF₃, —R⁹, —OR⁹, —C(═O)NR⁹R¹⁰, and        —C(═O)OR⁹, with the proviso that if the bond represented by a        dashed and solid line is a double bond then R³ and R⁵ are        absent.

In some embodiments, the compound of formula I has the structure offormula IVa, IVb, or IVc:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   the bond represented by a dashed and solid line represents a        bond selected from the group consisting of a single bond and a        double bond;    -   each R² and each R⁴ are independently selected from a group        consisting of Cl, F, CN, CF₃, —R⁹, —OR⁹, —C(═O)NR⁹R¹⁰, and        —C(═O)OR⁹; or alternatively, an R² and an R⁴ are taken together        with the atoms to which they are attached to form a substituted        or unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted carbocyclyl or substituted or        unsubstituted heterocyclyl;    -   each R³ and each R⁵ are independently selected from a group        consisting of Cl, F, CN, CF₃, —R⁹, —OR⁹, —C(═O)NR⁹R¹⁰, and        —C(═O)OR⁹, with the proviso that if the bond represented by a        dashed and solid line is a double bond then the R³ and R⁵        attached to the carbon atoms bonded that bond are absent.

Some embodiments include a pharmaceutical composition comprising atherapeutically effective amount of any one of the foregoing compoundsand a pharmaceutically acceptable excipient.

Some embodiments include any one of the foregoing compounds orcompositions for use in the treatment or prevention of a bacterialinfection.

Some embodiments include methods for treating or preventing a bacterialinfection comprising administering to a subject in need thereof, aneffective amount of any one of the foregoing compounds or compositions.

Some embodiments include the use of any one of the foregoing compoundsor compositions in the preparation of a medicament for the treatment orprevention of a bacterial infection.

Some embodiments further comprise administering an additionalmedicament, either is a separate composition or in the same composition.

In some embodiments, the additional medicament includes an antibacterialagent, antifungal agent, an antiviral agent, an anti-inflammatory agentor an anti-allergic agent.

In some embodiments, the additional medicament comprises anantibacterial agent such as a β-lactam.

In some embodiments, the β-lactam includes Amoxicillin, Ampicillin(Pivampicillin, Hetacillin, Bacampicillin, Metampicillin,Talampicillin), Epicillin, Carbenicillin (Carindacillin), Ticarcillin,Temocillin, Azlocillin, Piperacillin, Mezlocillin, Mecillinam(Pivmecillinam), Sulbenicillin, Benzylpenicillin (G), Clometocillin,Benzathine benzylpenicillin, Procaine benzylpenicillin, Azidocillin,Penamecillin, Phenoxymethylpenicillin (V), Propicillin, Benzathinephenoxymethylpenicillin, Pheneticillin, Cloxacillin (Dicloxacillin,Flucloxacillin), Oxacillin, Meticillin, Nafcillin, Faropenem, Biapenem,Doripenem, Ertapenem, Imipenem, Meropenem, Panipenem, Tomopenem,Razupenem, Cefazolin, Cefacetrile, Cefadroxil, Cefalexin, Cefaloglycin,Cefalonium, Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, Cefazedone,Cefazaflur, Cefradine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole,Cefminox, Cefonicid, Ceforanide, Cefotiam, Cefprozil, Cefbuperazone,Cefuroxime, Cefuzonam, Cefoxitin, Cefotetan, Cefmetazole, Loracarbef,Cefixime, Ceftazidime, Ceftriaxone, Cefcapene, Cefdaloxime, Cefdinir,Cefditoren, Cefetamet, Cefmenoxime, Cefodizime, Cefoperazone,Cefotaxime, Cefpimizole, Cefpiramide, Cefpodoxime, Cefsulodin, Cefteram,Ceftibuten, Cefatiolene, Ceftizoxime, Flomoxef, Latamoxef, Cefepime,Cefozopran, Cefpirome, Cefquinome, Ceftobiprole, Ceftaroline, CXA-101,RWJ-54428, MC-04,546, ME1036, BAL30072, SYN 2416, Ceftiofur, Cefquinome,Cefovecin, Aztreonam, Tigemonam, Carumonam, RWJ-442831, RWJ-333441, orRWJ-333442.

In some embodiments, the β-lactam includes Ceftazidime, Biapenem,Doripenem, Ertapenem, Imipenem, Meropenem, or Panipenem.

In some embodiments, the β-lactam is selected from Aztreonam, Tigemonam,BAL30072, SYN 2416, or Carumonam.

In some embodiments, the β-lactam Tigemonam, the composition is suitablefor oral administration, X is —CO₂R¹², and R¹² is selected from a groupconsisting of C₁₋₉alkyl, —(CH₂)₀₋₃—R₁₁, —C(R¹³)₂OC(O)C₁₋₉alkyl,—C(R¹³)₂OC(O)R¹¹, —C(R¹³)₂OC(O)OC₁₋₉alkyl and —C(R¹³)₂OC(O)OR¹¹.

In some embodiments, the infection that is treated or preventedcomprises a bacteria that includes Pseudomonas aeruginosa, Pseudomonasfluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes,Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia,Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii,Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi,Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri,Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes,Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens,Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteusvulgaris, Providencia alcalifaciens, Providencia rettgeri, Providenciastuartii, Acinetobacter baumannii, Acinetobacter calcoaceticus,Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis,Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis,Bordetella parapertussis, Bordetella bronchiseptica, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus,Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurellamultocida, Pasteurella haemolytica, Branhamella catarrhalis,Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni,Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrioparahaemolyticus, Legionella pneumophila, Listeria monocytogenes,Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella,Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis,Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroidesovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroideseggerthii, Bacteroides splanchnicus, Clostridium difficile,Mycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium leprae, Corynebacterium diphtheriae,Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcusagalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcusfaecium, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcushyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcushominis, or Staphylococcus saccharolyticus.

In some embodiments, the infection that is treated or preventedcomprises a bacteria that includes Pseudomonas aeruginosa, Pseudomonasfluorescens, Stenotrophomonas maltophilia, Escherichia coli, Citrobacterfreundii, Salmonella typhimurium, Salmonella typhi, Salmonellaparatyphi, Salmonella enteritidis, Shigella dysenteriae, Shigellaflexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes,Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens,Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersiniaenterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersiniaintermedia, Haemophilus influenzae, Haemophilus parainfluenzae,Haemophilus haemolyticus, Haemophilus parahaemolyticus, Helicobacterpylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli,Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila,Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis,Moraxella, Bacteroides fragilis, Bacteroides vulgatus, Bacteroidesovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroideseggerthii, or Bacteroides splanchnicus.

Some embodiments include a sterile container, comprising any one of theforegoing compounds in solid form and an antibacterial agent in solidform. In some embodiments, the antimicrobial agent is one of theadditional medicaments described above. Some embodiments include amethod of preparing a pharmaceutical composition for administration,comprising reconstituting the contents of the sterile container using apharmaceutically acceptable diluent. In some embodiments, thereconstituted solution is administered intravenously to a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the plasma concentration profile of a cyclicboronic acid ester derivative as a function of time after administrationto Sprague Dawley rats.

FIG. 2 is a graph depicting the plasma concentration profile of aprodrug of the cyclic boronic acid ester derivative of FIG. 1 as afunction of time after administration to Sprague Dawley rats.

FIG. 3 is an X-ray powder diffraction of a crystalline form of a cyclicboronic acid ester derivative.

FIG. 4 is a graph depicting an overlay of differential scanningcalorimetry and thermogravimetric results for the crystalline form ofFIG. 3.

DETAILED DESCRIPTION

The present invention relates to antimicrobial agents and potentiatorsthereof. Some embodiments include compounds, compositions,pharmaceutical compositions, uses thereof, including methods ofpreparation, and methods of treatment. In particular, the presentinvention relates to cyclic boronic acid ester derivatives. In someembodiments, the cyclic boronic acid ester derivatives have thestructure of formula I, II, IIIa, IIIb, IVa, IVb, or IVc as describedabove.

Some embodiments of the compound of formula II have the defined3,6-cis-stereochemistry shown in formula IIa:

or a pharmaceutically acceptable salt thereof.

Some embodiments of the compound of formula II have the defined3,6-trans-stereochemistry shown in formula IIb:

or a pharmaceutically acceptable salt thereof.

In one embodiment of the compound of formula II:

-   -   R¹ is selected from a group consisting of —C₁₋₉alkyl,        —C₂₋₉alkenyl, —C₂₋₉alkynyl, —NR⁹R¹⁰, —C₁₋₉alkylR¹¹,        —C₂₋₉alkenylR¹¹, —C₂₋₉alkynylR¹¹, —CH(OH)C₁₋₉alkylR⁹,        —CH(OH)C₂₋₉alkenylR⁹, —CH(OH)C₂₋₉alkynylR⁹, —C(═O)R⁹,        —C(═O)C₁₋₉alkylR⁹, —C(═O)C₂₋₉alkenylR⁹, —C(═O)C₂₋₉alkynylR⁹,        —C(═O)NR⁹R¹⁰, —N(R⁹)C(═O)R⁹, —N(R⁹)C(═O)NR⁹R¹⁰, —N(R⁹)C(═O)OR⁹,        —N(R⁹)C(═O)C(═NR¹⁰)R⁹, —N(R⁹)C(═O)C₁₋₄alkylN(R⁹)C(═O)R⁹,        —N(R⁹)C(═NR¹⁰)R⁹, —C(═NR¹⁰)NR⁹R¹⁰, —N═C(R⁹)NR⁹R¹⁰, —N(R⁹)SO₂R⁹,        —N(R⁹)SO₂NR⁹R¹⁰, substituted or unsubstituted aryl, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        carbocyclyl, and substituted or unsubstituted heterocyclyl;    -   R⁶ is selected from a group consisting of H, —C₁₋₉alkyl,        C₂₋₉alkenyl, —C₂₋₉alkynyl, —C₁₋₉alkylR¹¹, —C₂₋₉alkenylR¹¹,        —C₂₋₉alkynylR¹¹, —C(═O)OR⁹, and —C₁₋₉alkylCO₂R⁹,        —C₂₋₉alkenylCO₂R⁹, and —C₂₋₉alkynylCO₂R⁹, or alternatively R⁶        and an R⁷ are taken together with the atoms to which they are        attached to form a substituted or unsubstituted carbocyclyl or        substituted or unsubstituted heterocyclyl;    -   each R⁷ is independently selected from a group consisting of H,        —NR⁹R¹⁰, —OR⁹, and —C₁₋₉alkylCO₂R⁹, —C₂₋₉alkenylCO₂R⁹, and        —C₂₋₉alkynylCO₂R⁹, or independently, R⁶ and an R⁷ or        independently an R⁷ and an R⁸ are taken together with the atoms        to which they are attached to form a substituted or        unsubstituted carbocyclyl or substituted or unsubstituted        heterocyclyl;    -   each R⁸ is independently selected from a group consisting of H,        —NR⁹R¹⁰, —OR⁹, and —C₁₋₉alkylCO₂R⁹, —C₂₋₉alkenylCO₂R⁹, and        —C₂₋₉alkynylCO₂R⁹, or independently, an R⁷ and an R⁸ are taken        together with the atoms to which they are attached to form a        substituted or unsubstituted carbocyclyl or substituted or        unsubstituted heterocyclyl;    -   each R⁹ is independently selected from a group consisting of H,        —C₁₋₉alkyl, C₂₋₉alkenyl, —C₂₋₉alkynyl, —C₁₋₉alkylR¹¹,        —C₂₋₉alkenylR¹¹, —C₂₋₉alkynylR¹¹, substituted or unsubstituted        aryl, substituted or unsubstituted heteroaryl, substituted or        unsubstituted —(CH₂)₀₋₃carbocyclyl, and substituted or        unsubstituted heterocyclyl;    -   each R¹⁰ is independently selected from a group consisting of H,        —C₁₋₉alkyl, —OR⁹, —CH(═NH), substituted or unsubstituted aryl,        substituted or unsubstituted heteroaryl, substituted or        unsubstituted carbocyclyl, and substituted or unsubstituted        heterocyclyl; and    -   X is selected from a group consisting of H, —CO₂H and carboxylic        acid isosteres.

In some embodiments of compounds of formulas II, IIa, or IIb, n is 1.

In some embodiments of compounds of formulas II, IIa, or IIb, n is zero.

In some embodiments of compounds of formulas II, IIa, or IIb, n is 2.

Some embodiments of the compounds of formula IIIa or IIIb have the3,7-cis-stereochemistry shown in formula IIIc and IIId:

or a pharmaceutically acceptable salt thereof.

Some embodiments of the compounds of formula IIIa or IIIb have the3,7-trans-stereochemistry shown in formula IIIe and IIIf:

or a pharmaceutically acceptable salt thereof.

Some embodiments of the compounds of formulas IVa, IVb, or IVc have the3,8-cis-stereochemistry shown in formula IVd, IVe, and IVf:

or a pharmaceutically acceptable salt thereof.

Some embodiments of the compounds of formulas IVa, IVb, or IVc have the3,8-trans-stereochemistry shown in formula IVg, IVh, and IVi:

or a pharmaceutically acceptable salt thereof.

In some embodiments of the compounds of formulas II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, each R², R³, R⁴,and R⁵ are hydrogen.

In some embodiments of the compounds of formulas II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, at least one R²is substituted or unsubstituted aryl.

In some embodiments of the compounds of formulas II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, at least one R⁴is substituted or unsubstituted aryl.

In some embodiments of the compounds of formulas II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, at least one R²and R⁴ are taken together with the atoms to which they are attached toform a substituted or unsubstituted aryl.

In some embodiments of the compounds of formulas II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, the bondrepresented by a dashed and solid line is a single bond. In otherembodiments, the bond represented by a dashed and solid line is a doublebond.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R⁶ and each R⁷and R⁸ is hydrogen.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—NHC(═O)C₁₋₉alkylR¹¹. In some such embodiments, R¹¹ is substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl. In somesuch embodiments, R¹¹ is thien-2-yl.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—NHC(═O)C(═NOR⁹)R^(9′), wherein R^(9′) is selected from the groupconsisting of C₁₋₉alkyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted carbocyclyland substituted or unsubstituted heterocyclyl.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—NHC(═O)C₁₋₉alkylR¹¹. In some such embodiments, R¹¹ is substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted carbocyclyl, or substituted or unsubstitutedheterocyclyl.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—NHC(═O)R^(9′), wherein R^(9′) is substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcarbocyclyl, or substituted or unsubstituted heterocyclyl.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is —NR⁹R¹⁰.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—C₁₋₉alkylR¹¹.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—CH(OH)C₁₋₉alkylR⁹.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—C(═O)C₁₋₉alkylR⁹.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—C(═O)NR⁹R¹⁰.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—N(R⁸)C(═O)NR⁹R¹⁰.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—N(R⁹)C(═O)O R⁹.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—N(R⁹)C(═O)C₁₋₄alkylN(R⁹)C(═O)R⁹.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—N(R⁹)C(═NR¹⁰) R⁹.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—C(═NR¹⁰)NR⁹R¹⁰.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—N═C(R⁹)NR⁹R¹⁰.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—C(═O)C(═NR¹⁰) R⁹.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—N(R⁹)SO₂R⁹.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R¹ is—N(R⁹)SO₂NR⁹R¹⁰.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, X is —CO₂H.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, X is a carboxylicacid isostere. In some such embodiments, the carboxylic acid isostere isselected from the group consisting of —P(O)(OR⁹)₂, —P(O)(R⁹)(OR⁹),—P(O)(OR¹²)₂, —P(O)(R⁹)(OR¹²), —CON(R⁹)OH, —SO₃H, —SO₂N(R⁹)OH, and

wherein R^(12′) is selected from the group consisting of H, R₁₁,—C(R¹³)₂OC(O)C₁₋₉alkyl, —C(R¹³)₂OC(O)R¹¹, —C(R¹³)₂OC(O)OC₁₋₉alkyl andC(R¹³)₂OC(O)OR¹¹.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, m is 1.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R⁶, R⁷ and R⁸ areH.

In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb,IIIc, IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, and IVi, R⁷ is H; R⁸ is—C₁₋₉alkylCO₂R⁹; and R⁹ is H.

Some embodiments include a compound selected from the group consistingof:

or a pharmaceutically acceptable salt thereof.

Some embodiments include compounds selected from the group consistingof:

or a pharmaceutically acceptable salt thereof.

Definitions

Terms and substituents are given their ordinary meaning unless definedotherwise, and may be defined when introduced and retain theirdefinitions throughout unless otherwise specified, and retain theirdefinitions whether alone or as part of another group unless otherwisespecified.

As used herein, “alkyl” means a branched, or straight chain saturatedchemical group containing only carbon and hydrogen, such as methyl,isopropyl, isobutyl, sec-butyl and pentyl. In various embodiments, alkylgroups can either be unsubstituted or substituted with one or moresubstituents, e.g., halogen, hydroxyl, substituted hydroxyl, acyloxy,amino, substituted amino, amido, cyano, nitro, guanidino, amidino,mercapto, substituted mercapto, carboxy, sulfonyloxy, carbonyl,benzyloxy, aryl, heteroaryl, carbocyclyl, heterocyclyl, or otherfunctionality that may be suitably blocked with a protecting group.Typically, alkyl groups will comprise 1 to 20 carbon atoms, 1 to 9carbon atoms, preferably 1 to 6, and more preferably 1 to 5 carbonatoms.

As used herein, “alkenyl” means a straight or branched chain chemicalgroup containing only carbon and hydrogen and containing at least onecarbon-carbon double bond, such as 1-propenyl, 2-propenyl,2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. In variousembodiments, alkenyls can either be unsubstituted or substituted withone or more substituents, e.g., halogen, hydroxyl, substituted hydroxyl,acyloxy, amino, substituted amino, amido, cyano, nitro, guanidino,amidino, mercapto, substituted mercapto, carboxy, sulfonyloxy, carbonyl,benzyloxy, aryl, heteroaryl, carbocyclyl, heterocyclyl, or otherfunctionality that may be suitably blocked with a protecting group.Typically, alkenyl groups will comprise 2 to 20 carbon atoms, 2 to 9carbon atoms, preferably 2 to 6, and more preferably 2 to 5 carbonatoms.

As used herein, “alkynyl” means a straight or branched chain chemicalgroup containing only carbon and hydrogen and containing at least onecarbon-carbon triple bond, such as 1-propynyl, 1-butynyl, 2-butynyl, andthe like. In various embodiments, alkynyls can either be unsubstitutedor substituted with one or more substituents, e.g., halogen, hydroxyl,substituted hydroxyl, acyloxy, amino, substituted amino, amido, cyano,nitro, guanidino, amidino, mercapto, substituted mercapto, carboxy,sulfonyloxy, carbonyl, benzyloxy, aryl, heteroaryl, carbocyclyl,heterocyclyl, or other functionality that may be suitably blocked with aprotecting group. Typically, alkynyl groups will comprise 2 to 20 carbonatoms, 2 to 9 carbon atoms, preferably 2 to 6, and more preferably 2 to5 carbon atoms.

As used herein, “carbocyclyl” means a non-aromatic cyclic ring systemcontaining only carbon atoms in the ring system backbone, such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexenyl.Carbocyclyls may include multiple fused rings. Carbocyclyls may have anydegree of saturation provided that at least one ring in the ring systemis not aromatic. In various embodiments, carbocyclyl groups can eitherbe unsubstituted or substituted with one or more substituents, e.g.,halogen, alkoxy, acyloxy, amino, amido, cyano, nitro, hydroxyl,mercapto, carboxy, carbonyl, benzyloxy, aryl, heteroaryl, or otherfunctionality that may be suitably blocked with a protecting group.Typically, carbocyclyl groups will comprise 3 to 10 carbon atoms,preferably 3 to 6.

As used herein, “cycloalkyl” means a fully saturated carbocyclyl ringsystem. Examples include cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

As used herein, “cycloalkenyl” means a carbocyclyl ring system having atleast one double bond. An example is cyclohexenyl.

As used herein, “lower alkyl” means a subset of alkyl, and thus is ahydrocarbon substituent, which is linear, or branched. Preferred loweralkyls are of 1 to about 4 carbons, and may be branched or linear.Examples of lower alkyl include butyl, propyl, isopropyl, ethyl, andmethyl. Likewise, radicals using the terminology “lower” refer toradicals preferably with 1 to about 4 carbons in the alkyl portion ofthe radical.

As used herein, “aryl” means an aromatic radical having a single-ring(e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl)with only carbon atoms present in the ring backbone. In variousembodiments, aryl groups can either be unsubstituted or substituted withone or more substituents, e.g., amino, cyano, hydroxyl, lower alkyl,haloalkyl, alkoxy, nitro, halo, mercapto, carboxy, carbonyl, benzyloxy,aryl, heteroaryl, and other substituents. Some embodiments includesubstitution with an alkoxy group, which may be further substituted withone or more substituents, e.g., amino, cyano, hydroxyl, lower alkyl,haloalkyl, alkoxy, nitro, halo, mercapto, and other substituents. Apreferred aryl is phenyl.

As used herein, the term “heteroaryl” means an aromatic radical havingone or more heteroatom(s) (e.g., N, O, or S) in the ring backbone andmay include a single ring (e.g., pyridine) or multiple condensed rings(e.g., quinoline). In various embodiments, heteroaryl groups can eitherbe unsubstituted or substituted with one or more substituents, e.g.,amino, cyano, hydroxyl, lower alkyl, haloalkyl, alkoxy, nitro, halo,mercapto, carboxy, carbonyl, benzyloxy, aryl, heteroaryl, and othersubstituents. Examples of heteroaryl include thienyl, pyridyl, furyl,oxazolyl, oxadiazolyl, pyrollyl, imidazolyl, triazolyl, thiodiazolyl,pyrazolyl, isoxazolyl, thiadiazolyl, pyranyl, pyrazinyl, pyrimidinyl,pyridazinyl, triazinyl, thiazolyl, quinolinyl, quinazolinyl and others.

In these definitions it is contemplated that substitution on the aryland heteroaryl rings is within the scope of certain embodiments. Wheresubstitution occurs, the radical is called substituted aryl orsubstituted heteroaryl. Preferably one to three and more preferably oneor two substituents occur on the aryl ring. Though many substituentswill be useful, preferred substituents include those commonly found inaryl compounds, such as alkyl, cycloalkyl, hydroxy, alkoxy, cyano, halo,haloalkyl, mercapto and the like.

As used herein, “amide” or “amido” includes both RNR′CO— (in the case ofR=alkyl, alkylaminocarbonyl-) and RCONR′— (in the case of R=alkyl, alkylcarbonylamino-). “Amide” or “amido” includes a H—CON—, alkyl-CON—,carbocyclyl-CON—, aryl-CON—, heteroaryl-CON— or heterocyclyl-CON— group,wherein the alkyl, carbocyclyl, aryl or heterocyclyl group is as hereindescribed.

As used herein, the term “ester” includes both ROCO— (in the case ofR=alkyl, alkoxycarbonyl-) and RCOO— (in the case of R=alkyl,alkylcarbonyloxy-).

As used herein, “acyl” means an H—CO—, alkyl-CO—, carbocyclyl-CO—,aryl-CO—, heteroaryl-CO— or heterocyclyl-CO— group wherein the alkyl,carbocyclyl, aryl or heterocyclyl group is as herein described.Preferred acyls contain a lower alkyl. Exemplary alkyl acyl groupsinclude formyl, acetyl, propanoyl, 2-methylpropanoyl, t-butylacetyl,butanoyl and palmitoyl.

As used herein, “halo or halide” is a chloro, bromo, fluoro or iodo atomradical. Chloro and fluoro are preferred halides. The term “halo” alsocontemplates terms sometimes referred to as “halogen”, or “halide”.

As used herein, “heterocyclyl” means a non-aromatic cyclic ring systemcomprising at least one heteroatom in the ring system backbone.Heterocyclyls may include multiple fused rings. Heterocyclyls may haveany degree of saturation provided that at least one ring in the ringsystem is not aromatic. The heteroatom(s) may be present in either anon-aromatic or aromatic ring in the ring system. In variousembodiments, heterocyclyls may be substituted or unsubstituted with oneor more substituents, e.g., halogen, alkoxy, acyloxy, amino, amido,cyano, nitro, hydroxyl, mercapto, carboxy, carbonyl, benzyloxy, aryl,heteroaryl, and other substituents, and are attached to other groups viaany available valence, preferably any available carbon or nitrogen.Preferred heterocycles are of 5-7 members. In six membered monocyclicheterocycles, the heteroatom(s) are selected from one up to three of O,N or S, and when the heterocycle is five membered, preferably it has oneor two heteroatoms selected from O, N, or S. Examples of heterocyclylinclude pyrrolidinyl, piperidinyl, azepanyl, tetrahydrofuranyl,tetrahydropyranyl, oxepanyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, thiepanyl, indolinyl and dihydrobenzofuranyl.

As used herein, “substituted amino” means an amino radical which issubstituted by one or two alkyl, cycloalkyl, aryl, heteroaryl orheterocyclyl groups, wherein the alkyl, aryl, heteroaryl, cycloalkyl orheterocyclyl are defined as above.

As used herein, “substituted hydroxyl” means RO— group wherein R is analkyl, an aryl, heteroaryl, cycloalkyl or a heterocyclyl group, whereinthe alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl are defined asabove.

As used herein, “substituted thiol” means RS— group wherein R is analkyl, an aryl, heteroaryl, cycloalkyl or a heterocyclyl group, whereinthe alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl are defined asabove.

As used herein, “sulfonyl” means an alkylSO₂, arylSO₂, heteroarylSO₂,carbocyclylSO₂, or heterocyclyl-SO₂ group wherein the alkyl,carbocyclyl, aryl, heteroaryl or heterocyclyl are defined as above.

As used herein, “sulfamido” means an alkyl-N—S(O)₂N—, aryl-NS(O)₂N—,heteroaryl-NS(O)₂N—, carbocyclyl-NS(O)₂N or heterocyclyl-NS(O)₂N— groupwherein the alkyl, carbocyclyl, aryl, heteroaryl or heterocyclyl groupis as herein described.

As used herein, “sulfonamido” means an alkyl-S(O)₂N—, aryl-S(O)₂N—,heteroaryl-S(O)₂N—, carbocyclyl-S(O)₂N— or heterocyclyl-S(O)₂N— groupwherein the alkyl, carbocyclyl, aryl, heteroaryl or heterocyclyl groupis as herein described.

As used herein, “ureido” means an alkyl-NCON—, aryl-NCON—,heteroaryl-NCON—, carbocyclyl-NCON—, heterocyclyl-NCON— group orheterocyclyl-CON— group wherein the heterocyclyl group is attached by aring nitrogen, and wherein the alkyl, carbocyclyl, aryl, heteroaryl orheterocyclyl group is as herein described.

As used herein, “guanidino” means an alkyl-NC(═NR′)N—, aryl-NC(═NR′)N—,heteroaryl-NC(═NR′)N—, carbocyclyl-NC(═NR′)N— or heterocyclyl-NC(═NR′)N—group wherein R′ is an H, substituted or unsubstituted hydroxyl, CN,alkyl, aryl, heteroaryl or a heterocyclyl group, wherein the alkyl,carbocyclyl, aryl, heteroaryl or heterocyclyl group is as hereindescribed.

As used herein, a substituted group is derived from the unsubstitutedparent group in which there has been an exchange of one or more hydrogenatoms for another atom or group. When substituted, the substituentgroup(s) is (are) substituted with one or more substituent(s)individually and independently selected from C₁-C₆ alkyl, C₁-C₆ alkenyl,C₁-C₆ alkynyl, C₃-C₇ carbocycle (optionally substituted with halo,alkyl, alkoxy, carboxyl, haloalkyl, CN, —SO₂-alkyl, —CF₃, and —OCF₃),C₁-C₆ heteroalkyl, 5-7 membered heterocyclyl (e.g., tetrahydrofuryl)(optionally substituted with halo, alkyl, alkoxy, carboxyl, CN,—SO₂-alkyl, —CF₃, and —OCF₃), aryl (optionally substituted with halo,alkyl, aryl optionally substituted with C₁-C₆ alkyl, arylalkyl, alkoxy,carboxyl, CN, —SO₂-alkyl, —CF₃, and —OCF₃), arylalkyl (optionallysubstituted with halo, alkyl, alkoxy, aryl, carboxyl, CN, —SO₂-alkyl,—CF₃, and —OCF₃), heteroaryl (optionally substituted with halo, alkyl,alkoxy, aryl, aralkyl, carboxyl, CN, —SO₂-alkyl, —CF₃, and —OCF₃),heteroarylalkyl (optionally substituted with halo, alkyl, alkoxy, aryl,carboxyl, CN, —SO₂-alkyl, —CF₃, and —OCF₃), halo (e.g., chloro, bromo,iodo and fluoro), cyano, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxyalkyl (i.e.,ether), aryloxy, sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃),C₁-C₆ alkylthio, arylthio, amino (—NH₂), mono- and di-(C₁-C₆)alkylamino, quaternary ammonium salts, amino(C₁-C₆)alkoxy (e.g, —O(CH₂)₄NH₂),amino(C₁-C₆)alkoxyalkyl (e.g., —CH₂O(CH₂)₂NH₂),hydroxy(C₁-C₆)alkylamino, amino(C₁-C₆)alkylthio (e.g, —S(CH₂)₂NH₂),cyanoamino, nitro, carbamyl, oxo (═O), carboxy, glycolyl, glycyl,hydrazino, guanidinyl, sulfamyl, sulfonyl, sulfinyl, thiocarbonyl,thiocarboxy, C-amide, N-amide, N-carbamate, O-carbamate, and urea.Wherever a group is described as “optionally substituted” that group canbe substituted with the above substituents.

In some embodiments, substituted group(s) is (are) substituted with oneor more substituent(s) individually and independently selected fromC₁-C₆ alkyl, C₃-C₇ carbocycle, amino (—NH₂), amino(C₁-C₆)alkoxy,carboxyl, oxo (═O), C₁-C₆ alkylthio, amino(C₁-C₆)alkylthio, guanidinyl,aryl, 5-7 membered heterocyclyl, heteroarylalkyl, hydroxy, halo,amino(C₁-C₆)alkoxy, and amino(C₁-C₆)alkoxyalkyl.

In some embodiments, substituted group(s) is (are) substituted with oneor more substituent(s) individually and independently selected fromC₁-C₆ alkyl, amino (—NH₂), amino(C₁-C₆)alkoxy, carboxyl, oxo (═O), C₁-C₆alkylthio, amino(C₁-C₆)alkylthio, guanidinyl, hydroxy, halo,amino(C₁-C₆)alkoxy, and amino(C₁-C₆)alkoxyalkyl.

In some embodiments, substituted group(s) is (are) substituted with oneor more substituent(s) individually and independently selected fromC₁-C₆ alkyl, amino (—NH₂), carboxyl, oxo (═O), guanidinyl, hydroxy, andhalo.

It is to be understood that certain radical naming conventions caninclude either a mono-radical or a di-radical, depending on the context.For example, where a substituent requires two points of attachment tothe rest of the molecule, it is understood that the substituent is adi-radical. For example, a substituent identified as alkyl that requirestwo points of attachment includes di-radicals such as —CH₂—, —CH₂CH₂—,—CH₂CH(CH₃)CH₂—, and the like. Other radical naming conventions clearlyindicate that the radical is a di-radical. For example, as used herein,“alkylene” means a branched, or straight chain saturated di-radicalchemical group containing only carbon and hydrogen, such as methylene,isopropylene, isobutylene, sec-butylene, and pentylene, that is attachedto the rest of the molecule via two points of attachment. As usedherein, “alkenylene” means a straight or branched chain di-radicalchemical group containing only carbon and hydrogen and containing atleast one carbon-carbon double bond, such as 1-propenylene,2-propenylene, 2-methyl-1-propenylene, 1-butenylene, and 2-butenylene,that is attached to the rest of the molecule via two points ofattachment.

As used herein, “isosteres” of a chemical group are other chemicalgroups that exhibit the same or similar properties. For example,tetrazole is an isostere of carboxylic acid because it mimics theproperties of carboxylic acid even though they both have very differentmolecular formulae. Tetrazole is one of many possible isostericreplacements for carboxylic acid. Other carboxylic acid isosterescontemplated include —SO₃H, —SO₂HNR⁹, —PO₂(R⁹)₂, —PO₃(R⁹)₂,—CONHNHSO₂R⁹, —COHNSO₂R⁹, and —CONR⁹CN, where R⁹ is as defined above. Inaddition, carboxylic acid isosteres can include 5-7 membered carbocyclesor heterocycles containing any combination of CH₂, O, S, or N in anychemically stable oxidation state, where any of the atoms of said ringstructure are optionally substituted in one or more positions. Thefollowing structures are non-limiting examples of carbocyclic andheterocyclic isosteres contemplated. The atoms of said ring structuremay be optionally substituted at one or more positions with R⁹ asdefined above.

It is also contemplated that when chemical substituents are added to acarboxylic isostere, the compound retains the properties of a carboxylicisostere. It is contemplated that when a carboxylic isostere isoptionally substituted with one or more moieties selected from R⁹ asdefined above, then the substitution and substitution position isselected such that it does not eliminate the carboxylic acid isostericproperties of the compound. Similarly, it is also contemplated that theplacement of one or more R⁹ substituents upon a carbocyclic orheterocyclic carboxylic acid isostere is not a substitution at one ormore atom(s) that maintain(s) or is/are integral to the carboxylic acidisosteric properties of the compound, if such substituent(s) woulddestroy the carboxylic acid isosteric properties of the compound.

Other carboxylic acid isosteres not specifically exemplified in thisspecification are also contemplated.

The skilled artisan will recognize that some structures described hereinmay be resonance forms or tautomers of compounds that may be fairlyrepresented by other chemical structures, even when kinetically; theartisan recognizes that such structures are only a very small portion ofa sample of such compound(s). Such compounds are considered within thescope of the structures depicted, though such resonance forms ortautomers are not represented herein.

In some embodiments, due to the facile exchange of boron esters, thecompounds described herein may convert to or exist in equilibrium withalternate forms. Accordingly, in some embodiments, the compoundsdescribed herein may exist in combination with one or more of theseforms. For example, Compound 5 may exist in combination with one or moreopen-chain form (5a), dimeric form (5b), cyclic dimeric form (5c),trimeric form (5d), cyclic trimeric form (5e), and the like.

The compounds provided herein may encompass various stereochemicalforms. The compounds also encompasses diastereomers as well as opticalisomers, e.g. mixtures of enantiomers including racemic mixtures, aswell as individual enantiomers and diastereomers, which arise as aconsequence of structural asymmetry in certain compounds. Separation ofthe individual isomers or selective synthesis of the individual isomersis accomplished by application of various methods which are well knownto practitioners in the art.

The term “agent” or “test agent” includes any substance, molecule,element, compound, entity, or a combination thereof. It includes, but isnot limited to, e.g., protein, polypeptide, peptide or mimetic, smallorganic molecule, polysaccharide, polynucleotide, and the like. It canbe a natural product, a synthetic compound, or a chemical compound, or acombination of two or more substances. Unless otherwise specified, theterms “agent”, “substance”, and “compound” are used interchangeablyherein.

The term “analog” is used herein to refer to a molecule thatstructurally resembles a reference molecule but which has been modifiedin a targeted and controlled manner, by replacing a specific substituentof the reference molecule with an alternate substituent. Compared to thereference molecule, an analog would be expected, by one skilled in theart, to exhibit the same, similar, or improved utility. Synthesis andscreening of analogs, to identify variants of known compounds havingimproved characteristics (such as higher binding affinity for a targetmolecule) is an approach that is well known in pharmaceutical chemistry.

The term “mammal” is used in its usual biological sense. Thus, itspecifically includes humans, cattle, horses, dogs, cats, rats and micebut also includes many other species.

The term “microbial infection” refers to the invasion of the hostorganism, whether the organism is a vertebrate, invertebrate, fish,plant, bird, or mammal, by pathogenic microbes. This includes theexcessive growth of microbes that are normally present in or on the bodyof a mammal or other organism. More generally, a microbial infection canbe any situation in which the presence of a microbial population(s) isdamaging to a host mammal. Thus, a mammal is “suffering” from amicrobial infection when excessive numbers of a microbial population arepresent in or on a mammal's body, or when the effects of the presence ofa microbial population(s) is damaging the cells or other tissue of amammal. Specifically, this description applies to a bacterial infection.Note that the compounds of preferred embodiments are also useful intreating microbial growth or contamination of cell cultures or othermedia, or inanimate surfaces or objects, and nothing herein should limitthe preferred embodiments only to treatment of higher organisms, exceptwhen explicitly so specified in the claims.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. In addition, various adjuvants such as arecommonly used in the art may be included. These and other such compoundsare described in the literature, e.g., in the Merck Index, Merck &Company, Rahway, NJ. Considerations for the inclusion of variouscomponents in pharmaceutical compositions are described, e.g., in Gilmanet al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis ofTherapeutics, 8th Ed., Pergamon Press.

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of the compounds of thepreferred embodiments and, which are not biologically or otherwiseundesirable. In many cases, the compounds of the preferred embodimentsare capable of forming acid and/or base salts by virtue of the presenceof amino and/or carboxyl groups or groups similar thereto.Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids. Inorganic acids from which salts canbe derived include, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acidsfrom which salts can be derived include, for example, acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and thelike. Pharmaceutically acceptable base addition salts can be formed withinorganic and organic bases. Inorganic bases from which salts can bederived include, for example, sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum, and thelike; particularly preferred are the ammonium, potassium, sodium,calcium and magnesium salts. Organic bases from which salts can bederived include, for example, primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, basic ion exchange resins, and the like, specificallysuch as isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, and ethanolamine. Many such salts are known in the art,as described in WO 87/05297, Johnston et al., published Sep. 11, 1987(incorporated by reference herein in its entirety).

“Solvate” refers to the compound formed by the interaction of a solventand an EPI, a metabolite, or salt thereof. Suitable solvates arepharmaceutically acceptable solvates including hydrates.

“Subject” as used herein, means a human or a non-human mammal, e.g., adog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-humanprimate or a bird, e.g., a chicken, as well as any other vertebrate orinvertebrate.

A therapeutic effect relieves, to some extent, one or more of thesymptoms of the infection, and includes curing an infection. “Curing”means that the symptoms of active infection are eliminated, includingthe elimination of excessive members of viable microbe of those involvedin the infection. However, certain long-term or permanent effects of theinfection may exist even after a cure is obtained (such as extensivetissue damage).

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a pharmaceutical composition for prophylactic and/ortherapeutic purposes. The term “prophylactic treatment” refers totreating a patient who is not yet infected, but who is susceptible to,or otherwise at risk of, a particular infection, whereby the treatmentreduces the likelihood that the patient will develop an infection. Theterm “therapeutic treatment” refers to administering treatment to apatient already suffering from an infection.

Administration and Pharmaceutical Compositions

Some embodiments include pharmaceutical compositions comprising: (a) asafe and therapeutically effective amount of the cyclic boronic acidester derivative, or its corresponding enantiomer, diastereoisomer ortautomer, or pharmaceutically acceptable salt; and (b) apharmaceutically acceptable carrier.

The cyclic boronic acid ester derivatives are administered at atherapeutically effective dosage, e.g., a dosage sufficient to providetreatment for the disease states previously described. While humandosage levels have yet to be optimized for the compounds of thepreferred embodiments, generally, a daily dose for most of the cyclicboronic acid ester derivatives described herein is from about 0.25 mg/kgto about 120 mg/kg or more of body weight, from about 0.5 mg/kg or lessto about 70 mg/kg, from about 1.0 mg/kg to about 50 mg/kg of bodyweight, or from about 1.5 mg/kg to about 10 mg/kg of body weight. Thus,for administration to a 70 kg person, the dosage range would be fromabout 17 mg per day to about 8000 mg per day, from about 35 mg per dayor less to about 7000 mg per day or more, from about 70 mg per day toabout 6000 mg per day, from about 100 mg per day to about 5000 mg perday, or from about 200 mg to about 3000 mg per day. The amount of activecompound administered will, of course, be dependent on the subject anddisease state being treated, the severity of the affliction, the mannerand schedule of administration and the judgment of the prescribingphysician.

Administration of the compounds disclosed herein or the pharmaceuticallyacceptable salts thereof can be via any of the accepted modes ofadministration for agents that serve similar utilities including, butnot limited to, orally, subcutaneously, intravenously, intranasally,topically, transdermally, intraperitoneally, intramuscularly,intrapulmonary, vaginally, rectally, or intraocularly. Oral andparenteral administrations are customary in treating the indicationsthat are the subject of the preferred embodiments.

The compounds useful as described above can be formulated intopharmaceutical compositions for use in treatment of these conditions.Standard pharmaceutical formulation techniques are used, such as thosedisclosed in Remington's The Science and Practice of Pharmacy, 21st Ed.,Lippincott Williams & Wilkins (2005), incorporated by reference in itsentirety.

In addition to the selected compound useful as described above, comeembodiments include compositions containing apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier”, as used herein, means one or morecompatible solid or liquid filler diluents or encapsulating substances,which are suitable for administration to a mammal. The term“compatible”, as used herein, means that the components of thecomposition are capable of being commingled with the subject compound,and with each other, in a manner such that there is no interaction,which would substantially reduce the pharmaceutical efficacy of thecomposition under ordinary use situations. Pharmaceutically-acceptablecarriers must, of course, be of sufficiently high purity andsufficiently low toxicity to render them suitable for administrationpreferably to an animal, preferably mammal being treated.

Some examples of substances, which can serve aspharmaceutically-acceptable carriers or components thereof, are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents;flavoring agents; tableting agents, stabilizers; antioxidants;preservatives; pyrogen-free water; isotonic saline; and phosphate buffersolutions.

The choice of a pharmaceutically-acceptable carrier to be used inconjunction with the subject compound is basically determined by the waythe compound is to be administered.

The compositions described herein are preferably provided in unit dosageform. As used herein, a “unit dosage form” is a composition containingan amount of a compound that is suitable for administration to ananimal, preferably mammal subject, in a single dose, according to goodmedical practice. The preparation of a single or unit dosage formhowever, does not imply that the dosage form is administered once perday or once per course of therapy. Such dosage forms are contemplated tobe administered once, twice, thrice or more per day and may beadministered as infusion over a period of time (e.g., from about 30minutes to about 2-6 hours), or administered as a continuous infusion,and may be given more than once during a course of therapy, though asingle administration is not specifically excluded. The skilled artisanwill recognize that the formulation does not specifically contemplatethe entire course of therapy and such decisions are left for thoseskilled in the art of treatment rather than formulation.

The compositions useful as described above may be in any of a variety ofsuitable forms for a variety of routes for administration, for example,for oral, nasal, rectal, topical (including transdermal), ocular,intracerebral, intracranial, intrathecal, intra-arterial, intravenous,intramuscular, or other parental routes of administration. The skilledartisan will appreciate that oral and nasal compositions comprisecompositions that are administered by inhalation, and made usingavailable methodologies. Depending upon the particular route ofadministration desired, a variety of pharmaceutically-acceptablecarriers well-known in the art may be used. Pharmaceutically-acceptablecarriers include, for example, solid or liquid fillers, diluents,hydrotropies, surface-active agents, and encapsulating substances.Optional pharmaceutically-active materials may be included, which do notsubstantially interfere with the inhibitory activity of the compound.The amount of carrier employed in conjunction with the compound issufficient to provide a practical quantity of material foradministration per unit dose of the compound. Techniques andcompositions for making dosage forms useful in the methods describedherein are described in the following references, all incorporated byreference herein: Modem Pharmaceutics, 4th Ed., Chapters 9 and 10(Banker & Rhodes, editors, 2002); Lieberman et al., PharmaceuticalDosage Forms: Tablets (1989); and Ansel, Introduction to PharmaceuticalDosage Forms 8th Edition (2004).

Various oral dosage forms can be used, including such solid forms astablets, capsules, granules and bulk powders. These oral forms comprisea safe and effective amount, usually at least about 5%, with a maximumof about 90%, of the compound. Tablets can be compressed, tablettriturates, enteric-coated, sugar-coated, film-coated, ormultiple-compressed, containing suitable binders, lubricants, diluents,disintegrating agents, coloring agents, flavoring agents, flow-inducingagents, and melting agents. Liquid oral dosage forms include aqueoussolutions, emulsions, suspensions, solutions and/or suspensionsreconstituted from non-effervescent granules, and effervescentpreparations reconstituted from effervescent granules, containingsuitable solvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, melting agents, coloring agents and flavoringagents.

The pharmaceutically-acceptable carrier suitable for the preparation ofunit dosage forms for peroral administration is well-known in the art.Tablets typically comprise conventional pharmaceutically-compatibleadjuvants as inert diluents, such as calcium carbonate, sodiumcarbonate, mannitol, lactose and cellulose; binders such as starch,gelatin and sucrose; disintegrants such as starch, alginic acid andcroscarmellose; lubricants such as magnesium stearate, stearic acid andtalc. Glidants such as silicon dioxide can be used to improve flowcharacteristics of the powder mixture. Coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. The selection of carriercomponents depends on secondary considerations like taste, cost, andshelf stability, which are not critical, and can be readily made by aperson skilled in the art.

Peroral compositions also include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically-acceptable carrierssuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, AVICEL RC-591, tragacanth and sodium alginate; typicalwetting agents include lecithin and polysorbate 80; and typicalpreservatives include methyl paraben and sodium benzoate. Peroral liquidcompositions may also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

Such compositions may also be coated by conventional methods, typicallywith pH or time-dependent coatings, such that the subject compound isreleased in the gastrointestinal tract in the vicinity of the desiredtopical application, or at various times to extend the desired action.Such dosage forms typically include, but are not limited to, one or moreof cellulose acetate phthalate, polyvinylacetate phthalate,hydroxypropyl methyl cellulose phthalate, ethyl cellulose, Eudragitcoatings, waxes and shellac.

Compositions described herein may optionally include other drug actives.

Other compositions useful for attaining systemic delivery of the subjectcompounds include sublingual, buccal and nasal dosage forms. Suchcompositions typically comprise one or more of soluble filler substancessuch as sucrose, sorbitol and mannitol; and binders such as acacia,microcrystalline cellulose, carboxymethyl cellulose and hydroxypropylmethyl cellulose. Glidants, lubricants, sweeteners, colorants,antioxidants and flavoring agents disclosed above may also be included.

A liquid composition, which is formulated for topical ophthalmic use, isformulated such that it can be administered topically to the eye. Thecomfort should be maximized as much as possible, although sometimesformulation considerations (e.g. drug stability) may necessitate lessthan optimal comfort. In the case that comfort cannot be maximized, theliquid should be formulated such that the liquid is tolerable to thepatient for topical ophthalmic use. Additionally, an ophthalmicallyacceptable liquid should either be packaged for single use, or contain apreservative to prevent contamination over multiple uses.

For ophthalmic application, solutions or medicaments are often preparedusing a physiological saline solution as a major vehicle. Ophthalmicsolutions should preferably be maintained at a comfortable pH with anappropriate buffer system. The formulations may also containconventional, pharmaceutically acceptable preservatives, stabilizers andsurfactants.

Preservatives that may be used in the pharmaceutical compositionsdisclosed herein include, but are not limited to, benzalkonium chloride,PHMB, chlorobutanol, thimerosal, phenylmercuric, acetate andphenylmercuric nitrate. A useful surfactant is, for example, Tween 80.Likewise, various useful vehicles may be used in the ophthalmicpreparations disclosed herein. These vehicles include, but are notlimited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose,poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose and purifiedwater.

Tonicity adjustors may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, mannitol and glycerin, or any other suitable ophthalmicallyacceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as theresulting preparation is ophthalmically acceptable. For manycompositions, the pH will be between 4 and 9. Accordingly, buffersinclude acetate buffers, citrate buffers, phosphate buffers and boratebuffers. Acids or bases may be used to adjust the pH of theseformulations as needed.

In a similar vein, an ophthalmically acceptable antioxidant includes,but is not limited to, sodium metabisulfite, sodium thiosulfate,acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.

Other excipient components, which may be included in the ophthalmicpreparations, are chelating agents. A useful chelating agent is edetatedisodium, although other chelating agents may also be used in place orin conjunction with it.

For topical use, creams, ointments, gels, solutions or suspensions,etc., containing the compound disclosed herein are employed. Topicalformulations may generally be comprised of a pharmaceutical carrier,co-solvent, emulsifier, penetration enhancer, preservative system, andemollient.

For intravenous administration, the compounds and compositions describedherein may be dissolved or dispersed in a pharmaceutically acceptablediluent, such as a saline or dextrose solution. Suitable excipients maybe included to achieve the desired pH, including but not limited toNaOH, sodium carbonate, sodium acetate, HCl, and citric acid. In variousembodiments, the pH of the final composition ranges from 2 to 8, orpreferably from 4 to 7. Antioxidant excipients may include sodiumbisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate,thiourea, and EDTA. Other non-limiting examples of suitable excipientsfound in the final intravenous composition may include sodium orpotassium phosphates, citric acid, tartaric acid, gelatin, andcarbohydrates such as dextrose, mannitol, and dextran. Furtheracceptable excipients are described in Powell, et al., Compendium ofExcipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998,52 238-311 and Nema et al., Excipients and Their Role in ApprovedInjectable Products: Current Usage and Future Directions, PDA J PharmSci and Tech 2011, 65 287-332, both of which are incorporated herein byreference in their entirety. Antimicrobial agents may also be includedto achieve a bacteriostatic or fungistatic solution, including but notlimited to phenylmercuric nitrate, thimerosal, benzethonium chloride,benzalkonium chloride, phenol, cresol, and chlorobutanol.

The resulting composition may be infused into the patient over a periodof time. In various embodiments, the infusion time ranges from 5 minutesto continuous infusion, from 10 minutes to 8 hours, from 30 minutes to 4hours, and from 1 hour to 3 hours. In one embodiment, the drug isinfused over a 3 hour period. The infusion may be repeated at thedesired dose interval, which may include, for example, 6 hours, 8 hours,12 hours, or 24 hours.

The compositions for intravenous administration may be provided tocaregivers in the form of one more solids that are reconstituted with asuitable diluent such as sterile water, saline or dextrose in watershortly prior to administration. Reconstituted concentrated solutionsmay be further diluted into a parenteral solutions having a volume offrom about 25 to about 1000 ml, from about 30 ml to about 500 ml, orfrom about 50 ml to about 100 ml. In other embodiments, the compositionsare provided in solution ready to administer parenterally. In stillother embodiments, the compositions are provided in a solution that isfurther diluted prior to administration. In embodiments that includeadministering a combination of a compound described herein and anotheragent, the combination may be provided to caregivers as a mixture, orthe caregivers may mix the two agents prior to administration, or thetwo agents may be administered separately.

The actual dose of the active compounds described herein depends on thespecific compound, and on the condition to be treated; the selection ofthe appropriate dose is well within the knowledge of the skilledartisan.

Kits for Intravenous Administration

Some embodiments include a kit comprising a compound described hereinand an additional agent, such as an antimicrobial agent. In oneembodiment, both components are provided in a single sterile container.In the case of solids for reconstitution, the agents may be pre-blendedand added to the container simultaneously or may be dry-powder filledinto the container in two separate steps. In some embodiments, thesolids are sterile crystalline products. In other embodiment, the solidsare lyophiles. In one embodiment, both components are lyophilizedtogether. Non-limiting examples of agents to aid in lyophilizationinclude sodium or potassium phosphates, citric acid, tartaric acid,gelatin, and carbohydrates such as dextrose, mannitol, and dextran. Oneembodiment includes non-sterile solids that are irradiated either beforeor after introduction into the container.

In the case of a liquid, the agents may be dissolved or dispersed in adiluent ready for administration. In another embodiment, the solution ordispersion may be further diluted prior to administration. Someembodiments include providing the liquid in an IV bag. The liquid may befrozen to improve stability.

In one embodiment, the container includes other ingredients such as a pHadjuster, a solubilizing agent, or a dispersing agent. Non-limitingexamples of pH adjusters include NaOH, sodium carbonate, sodium acetate,HCl, and citric acid.

The molar ratio of compound described herein to additional agent (e.g.,antibacterial agent) may be from about 10:1 to 1:10, 8:1 to 1:8, 5:1 to1:5, 3:1 to 1:3, 2:1 to 1:2, or about 1:1. In various embodiments theamount of compound described herein may be from 100 mg to 5 g, 500 mg to2 g, or about 1 g. Similarly, in various embodiments the amount ofadditional agent may be from 100 mg to 5 g, 500 mg to 2 g, or about 1 g.

In an alternative embodiment, the two components may be provided inseparate containers. Each container may include a solid, solution, ordispersion. In such embodiments, the two containers may be provided in asingle package or may be provided separately. In one embodiment, thecompound described herein is provided as a solution while the additionalagent (e.g., antibacterial agent) is provided as a solid ready forreconstitution. In one such embodiment, the solution of the compounddescribed herein is used as the diluent to reconstitute the other agent.

Methods of Treatment

Some embodiments of the present invention include methods of treatingbacterial infections with the compounds and compositions comprisingcyclic boronic acid ester derivatives described herein. Some methodsinclude administering a compound, composition, pharmaceuticalcomposition described herein to a subject in need thereof. In someembodiments, a subject can be an animal, e.g., a mammal, a human. Insome embodiments, the bacterial infection comprises a bacteria describedherein. As will be appreciated from the foregoing, methods of treating abacterial infection include methods for preventing bacterial infectionin a subject at risk thereof.

Further embodiments include administering a combination of compounds toa subject in need thereof. A combination can include a compound,composition, pharmaceutical composition described herein with anadditional medicament.

Some embodiments include co-administering a compound, composition,and/or pharmaceutical composition described herein, with an additionalmedicament. By “co-administration,” it is meant that the two or moreagents may be found in the patient's bloodstream at the same time,regardless of when or how they are actually administered. In oneembodiment, the agents are administered simultaneously. In one suchembodiment, administration in combination is accomplished by combiningthe agents in a single dosage form. When combining the agents in asingle dosage form, they may be physically mixed (e.g, by co-dissolutionor dry mixing) or may form an adduct or be covalently linked such thatthey split into the two or more active ingredients upon administrationto the patient. In another embodiment, the agents are administeredsequentially. In one embodiment the agents are administered through thesame route, such as orally. In another embodiment, the agents areadministered through different routes, such as one being administeredorally and another being administered i.v.

Examples of additional medicaments include an antibacterial agent,antifungal agent, an antiviral agent, an anti-inflammatory agent and ananti-allergic agent.

Some embodiments include co-administration of a compound, composition orpharmaceutical composition described herein with an antibacterial agentsuch as a β-lactam. Examples of such β-lactams include Amoxicillin,Ampicillin (e.g., Pivampicillin, Hetacillin, Bacampicillin,Metampicillin, Talampicillin), Epicillin, Carbenicillin (Carindacillin),Ticarcillin, Temocillin, Azlocillin, Piperacillin, Mezlocillin,Mecillinam (Pivmecillinam), Sulbenicillin, Benzylpenicillin (G),Clometocillin, Benzathine benzylpenicillin, Procaine benzylpenicillin,Azidocillin, Penamecillin, Phenoxymethylpenicillin (V), Propicillin,Benzathine phenoxymethylpenicillin, Pheneticillin, Cloxacillin (e.g.,Dicloxacillin, Flucloxacillin), Oxacillin, Methicillin, Nafcillin,Faropenem, Biapenem, Doripenem, Ertapenem, Imipenem, Meropenem,Panipenem, Tomopenem, Razupenem, Cefazolin, Cefacetrile, Cefadroxil,Cefalexin, Cefaloglycin, Cefalonium, Cefaloridine, Cefalotin, Cefapirin,Cefatrizine, Cefazedone, Cefazaflur, Cefradine, Cefroxadine, Ceftezole,Cefaclor, Cefamandole, Cefminox, Cefonicid, Ceforanide, Cefotiam,Cefprozil, Cefbuperazone, Cefuroxime, Cefuzonam, Cefoxitin, Cefotetan,Cefmetazole, Loracarbef, Cefixime, Ceftazidime, Ceftriaxone, Cefcapene,Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefmenoxime, Cefodizime,Cefoperazone, Cefotaxime, Cefpimizole, Cefpiramide, Cefpodoxime,Cefsulodin, Cefteram, Ceftibuten, Cefatiolene, Ceftizoxime, Flomoxef,Latamoxef, Cefepime, Cefozopran, Cefpirome, Cefquinome, Ceftobiprole,Ceftaroline, CXA-101, RWJ-54428, MC-04,546, ME1036, BAL30072, SYN 2416,Ceftiofur, Cefquinome, Cefovecin, Aztreonam, Tigemonam, Carumonam,RWJ-442831, RWJ-333441, and RWJ-333442.

Preferred embodiments include β-lactams such as Ceftazidime, Biapenem,Doripenem, Ertapenem, Imipenem, Meropenem, ME1036, Tomopenem, Razupenem,and Panipenem.

Some embodiments include co-administration of the compounds,compositions and/or pharmaceutical compositions described herein with anadditional agent, wherein the additional agent comprises a monobactam.Examples of monobactams include aztreonam, tigemonam, BAL 30072, SYN2416 (BAL19764), and carumonam.

Some embodiments include co-administration of the compounds,compositions and/or pharmaceutical compositions described herein with anadditional agent, wherein the additional agent comprises a Class A, B,C, or D beta-lactamase inhibitor. An example of a class B beta lactamaseinhibitor includes ME1071 (Yoshikazu Ishii et al, “In Vitro Potentiationof Carbapenems with ME1071, a Novel Metallo-1-Lactamase Inhibitor,against Metallo-1-lactamase Producing Pseudomonas aeruginosa ClinicalIsolates.” Antimicrob. Agents Chemother. doi:10.1128/AAC.01397-09 (July2010)). Other examples of beta-lactamase inhibitors administered as anadditional agent include clavulanic acid, tazobactam, sulbactam,avibactam (NXL-104), MK-7655, and BAL29880. MK-7655 has the followingstructure:

Indications

The compounds and compositions comprising cyclic boronic acid esterderivatives described herein can be used to treat bacterial infections.Bacterial infections that can be treated with the compounds,compositions and methods described herein can comprise a wide spectrumof bacteria. Example organisms include gram-positive bacteria,gram-negative bacteria, aerobic and anaerobic bacteria, such asStaphylococcus, Lactobacillus, Streptococcus, Sarcina, Escherichia,Enterobacter, Klebsiella, Pseudomonas, Acinetobacter, Mycobacterium,Proteus, Campylobacter, Citrobacter, Neisseria, Bacillus, Bacteroides,Peptococcus, Clostridium, Salmonella, Shigella, Serratia, Haemophilus,Brucella and other organisms.

More examples of bacterial infections include Pseudomonas aeruginosa,Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonasalcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia,Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli,Citrobacter freundii, Salmonella typhimurium, Salmonella typhi,Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae,Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacteraerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratiamarcescens, Francisella tularensis, Morganella morganii, Proteusmirabilis, Proteus vulgaris, Providencia alcalifaciens, Providenciarettgeri, Providencia stuartii, Acinetobacter baumannii, Acinetobactercalcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica,Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia,Bordetella pertussis, Bordetella parapertussis, Bordetellabronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae,Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilusducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamellacatarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacterjejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae,Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes,Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella,Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis,Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroidesovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroideseggerthii, Bacteroides splanchnicus, Clostridium difficile,Mycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium leprae, Corynebacterium diphtheriae,Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcusagalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcusfaecium, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcushyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcushominis, or Staphylococcus saccharolyticus.

The following examples will further describe the present invention, andare used for the purposes of illustration only, and should not beconsidered as limiting.

EXAMPLES General Procedures

Materials used in preparing the cyclic boronic acid ester derivativesdescribed herein may be made by known methods or are commerciallyavailable. It will be apparent to the skilled artisan that methods forpreparing precursors and functionality related to the compounds claimedherein are generally described in the literature including, for example,procedures described in U.S. Pat. No. 7,271,186 and WO2009064414, eachof which is incorporated by reference in its entirety. In thesereactions, it is also possible to make use of variants which arethemselves known to those of ordinary skill in this art, but are notmentioned in greater detail. The skilled artisan given the literatureand this disclosure is well equipped to prepare any of the compounds.

It is recognized that the skilled artisan in the art of organicchemistry can readily carry out manipulations without further direction,that is, it is well within the scope and practice of the skilled artisanto carry out these manipulations. These include reduction of carbonylcompounds to their corresponding alcohols, oxidations, acylations,aromatic substitutions, both electrophilic and nucleophilic,etherifications, esterification and saponification and the like. Thesemanipulations are discussed in standard texts such as March AdvancedOrganic Chemistry (Wiley), Carey and Sundberg, Advanced OrganicChemistry (incorporated herein by reference in its entirety) and thelike.

The skilled artisan will readily appreciate that certain reactions arebest carried out when other functionality is masked or protected in themolecule, thus avoiding any undesirable side reactions and/or increasingthe yield of the reaction. Often the skilled artisan utilizes protectinggroups to accomplish such increased yields or to avoid the undesiredreactions. These reactions are found in the literature and are also wellwithin the scope of the skilled artisan. Examples of many of thesemanipulations can be found for example in T. Greene and P. WutsProtecting Groups in Organic Synthesis, 4th Ed., John Wiley & Sons(2007), incorporated herein by reference in its entirety.

The following example schemes are provided for the guidance of thereader, and represent preferred methods for making the compoundsexemplified herein. These methods are not limiting, and it will beapparent that other routes may be employed to prepare these compounds.Such methods specifically include solid phase based chemistries,including combinatorial chemistry. The skilled artisan is thoroughlyequipped to prepare these compounds by those methods given theliterature and this disclosure. The compound numberings used in thesynthetic schemes depicted below are meant for those specific schemesonly, and should not be construed as or confused with same numberings inother sections of the application.

Trademarks used herein are examples only and reflect illustrativematerials used at the time of the invention. The skilled artisan willrecognize that variations in lot, manufacturing processes, and the like,are expected. Hence the examples, and the trademarks used in them arenon-limiting, and they are not intended to be limiting, but are merelyan illustration of how a skilled artisan may choose to perform one ormore of the embodiments of the invention.

(¹H) nuclear magnetic resonance spectra (NMR) were measured in theindicated solvents on either a Bruker NMR spectrometer (Avance™ DRX500,500 MHz for 1H) or Varian NMR spectrometer (Mercury 400BB, 400 MHz for1H). Peak positions are expressed in parts per million (ppm) downfieldfrom tetramethylsilane. The peak multiplicities are denoted as follows,s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sex,sextet; sep, septet; non, nonet; dd, doublet of doublets; td, triplet ofdoublets; m, multiplet.

The following abbreviations have the indicated meanings:

-   -   n-BuLi=n-butyllithium    -   t-Bu=tert-butyl    -   DCM=dichloromethane    -   DMF=N,N-dimethylformamide    -   DIPEA=diisopropylethylamine    -   EDCI=1-ethyl-3-(3-dimethylaminopropyl) carbodiimide    -   ESBL=extended-spectrum β-lactamase    -   ESIMS=electron spray mass spectrometry    -   EtOAc=ethyl acetate    -   EtOH=ethanol    -   HATU=2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HCl=hydrochloric acid    -   HOBt=hydroxybenzotriazole    -   Im=imidazole    -   LiHMDS=lithium bis(trimethylsilyl)amide    -   MeCN=acetonitrile    -   NaHCO₃=sodium bicarbonate    -   Na₂SO₄=sodium sulfate    -   NMM=N-methylmorpholine    -   NMR=nuclear magnetic resonance    -   Pd/C=palladium on carbon    -   TBDMSCl=tert-butyldimethylsilyl chloride    -   TBS=tert-butyldimethylsilyl    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran    -   TLC=thin layer chromatography    -   TMS=trimethylsilyl    -   TPPB=tris(pentafluorophenyl)borane monohydrate

The following example schemes are provided for the guidance of thereader, and collectively represent an example method for making thecompounds provided herein. Furthermore, other methods for preparingcompounds described herein will be readily apparent to the person ofordinary skill in the art in light of the following reaction schemes andexamples. Unless otherwise indicated, all variables are as definedabove.

Compounds of formula I where R¹ is an acylamino group and X is acarboxylic acid can be prepared as depicted in Scheme 1.

The addition of enolates to substituted α,β-unsaturated ketones oraldehydes to form β-hydroxy esters is a well-known reaction (Scheme 1).Substituents R⁷ and R⁸ of formula I may be controlled by use of theappropriate α-mono or di-substituted ester III. Similarly, substituentsR², R³, and R⁴ may be controlled by use of the appropriate substitutedsubstituted α,β-unsaturated ketones or aldehydes analog II. Precursorsof structure IV, where R⁶ and R⁷ or R⁸ are combined together, may bemade following the known procedures [J. Am. Chem. Soc. (1982), 104,1735-7, Tetrahedron Lett. (2003), 44, 1259-62]. The β-hydroxy ester ofstructure IV is protected with an acid-sensitive protecting group,affording V; this selection allows simultaneous deprotection of theboronate ester and hydroxyl protecting group in the final step,resulting in a cyclized product. The pinacol boronate VII is formed fromsubstituted V using iridium catalysis [Tetrahedron (2004), 60,10695-700]. Trans-esterification was readily achieved with opticallyactive pinane diol VIII to result in IX [Tetrahedron: Asymmetry, (1997),8, 1435-40]. Transesterification may also be achieved from the catecholester analog of VII. Such catechol esters can be made by reaction of Vwith commercially available catechol borane [Tetrahedron (1989), 45,1859-85]. Homologation of IX to give chloromethylene addition product Xwith good stereocontrol may be achieved via Matteson reaction conditions(WO00946098). The chloro derivative X can be utilized to introduce asubstituted amine group at the C3-position of the oxaborinane-2-ol.Stereospecific substitution with hexamethyldisilazane gives thecorresponding bis(trimethylsilyl) amide XI which may be reacted in situwith an acid chloride to result directly in analogs of structure XII.Such analogs of XII can also be made via coupling of the bis-TMS aminewith commercially available carboxylic acids under typical amidecoupling conditions (e.g., carbodiimide or HATU coupling). Compounds ofFormula 1 where R¹ is substituted with —N(R⁹)C(═O)C(═NOR⁹)R⁹ may besynthesized from corresponding carboxylic acids via coupling of XI toXII as in scheme 1. Such carboxylic acids can be made by following theprocedures described U.S. Pat. No. 5,888,998, U.S. ApplicationPublication No. 2004/0019203, and U.S. Pat. No. 4,822,786, all of whichare incorporated herein by reference in their entirety. Simultaneousdeprotection of the pinane ester, the tert-butyldimethylsilyloxy groupand the tert-butyl ester group and concomitant cyclization are achievedby heating with dilute HCl, affording the desired oxaborinanederivatives of structure XIII. This transformation may also be achievedby treatment with BCl₃ or BBr3. Alternatively, the deprotection may beattained via trans-esterification with isobutyl boronic acid in presenceof dilute HCl (WO009064413).

Compounds of structure XVI where R¹ of Formula I is an alkyl, aralkyl oraminoaryl group may be made from bromo intermediate XIV as shown inScheme 2 [J. Organomet. Chem. (1992), 431, 255-70]. Such bromoderivatives may be made as analogously to the chloro compounds of Scheme1, utilizing dibromomethane [J. Am. Chem. Soc. (1990), 112, 3964-969].Displacement of the bromo group in XIV can be achieved by α-alkoxysubstituted alkyllithium agents [J. Am. Chem. Soc. (1989), 111,4399-402; J. Am. Chem. Soc. (1988), 110, 842-53] or organomagnesiumreagents (WO00946098) or by the sodium salt of alkyl or aryl carbamatederivatives [J. Org. Chem. (1996), 61, 7951-54], resulting in XV.Cyclization of XV to afford XVI may be achieved under the conditionsdescribed in Scheme 1.

Compounds of formula XIII and XVI are mixtures of 3,6-cis- and3,6-trans-isomers. These analogs can be made in enantiomerically pureform as single isomers by starting (as in Scheme 1) with a singleenantiomer (XVII), as shown in Scheme 3. A variety of methods to preparesuch enantiomerically pure β-hydroxy esters are known in literature, forexample via resolution [Org. Lett., (2008), 10, 3907-09] orstereoselective synthesis [Tetrahedron, (2000), 56, 917-47]. Such singleisomers result in enantiomerically pure cis-compounds XIII or XVI whenused in the sequences depicted in Schemes 1 and 2.

The sequence shown in Scheme 1 also allows for varied ring sizes informula I such as 7- and 8-membered rings. For example, a seven-memberedanalog XX where n=1 can be achieved by using the corresponding allylintermediate (XIX) as a starting material (Scheme 4). Such allylderivatives as XIX can be made utilizing one of several well knownβ-hydroxy ester preparations [Tetrahedron (2007), 63, 8336-50].Intermediate XIX where n=2 can be prepared as described in Scheme 1 togive corresponding 8-membered compound of structure XX starting frompent-4-ene-1-al [J. Med. Chem. (1998), 41(6), 965-972].

Compounds of formula XXVI and XXVII can be made following the sequencedepicted in Scheme 5. Ring-Closing Metathesis reaction with boronatedolefins (XXI) and olefin substituted β-hydroxy esters (XXII) result incyclic boronates of formula XXIII. Such cyclic boronates (XXIII) undergoready esterification with (+)-pinane diol to give required Mattesonreaction precursors upon protection of the resulting alcohol with groupssuch as t-butyldimethylsilyl- or benzyl or trityl. Matteson homologationfollowed by amide formation result in compounds of formula XXV with highstereoselectivity, as described above. Acid mediated hydrolysis ofcompounds of XXV upon deprotection give cyclic boronate (XXVI). Doublebond substitution of XXVI can be further modified to other analogs suchas saturated cyclic boronate (XXVII) by catalytic hydrogenation. Theabove sequence can be utilized to make 7- or 8-membered rings withdouble bond at a desired position by varying p and q of XXI and XXII.

Compounds of formula I where R² and R⁴ taken together form an aryl ringcan be made from commercially available substituted aryl precursors asXXVIII. Substitution of the bromine atom by a boronate ester may be doneunder palladium catalyzed conditions [Tetrahedron (2002), 58, 9633-95].The steps of hydroxy ester formation, α-amidoboronate preparation andcyclization can be attained by synthetic steps analogous to those inScheme 1 to give compounds XXIX.

Compounds of formula I where R⁷ and R⁸ are substituted as maleate (XXXV)or succinate (XXXVI) may be made following the sequence shown in Scheme7. Maleate intermediates such as XXXII can be transformed to analogsXXXV analogously to the steps in Scheme 1. Analogs of XXXV can befurther transformed to the corresponding succinic acids of structureXXXVI by catalytic hydrogenation. Maleate intermediate XXXII may beassembled from intermediate XXXI by successive deprotection of the TBSgroup, oxidation to the aldehyde, addition of vinyl Grignard andreprotection as a TBS ether. Intermediate XXXI may be formed from aprotected propargylic alcohol XXX following methods known in theliterature [Tetrahedron, (2002), 58, 6545-54].

Compounds of Formula I where X is a carboxylic acid isostere can beprepared following the protocols described in the literature (see J.Med. Chem. 2011, 54, 2529-2591, which is incorporated herein byreference in its entirety).

Illustrative Compound Examples

Synthesis of2-((3R)-2-hydroxy-3-(2-(thiophen-2-yl)acetamido)-1,2-oxaborinan-6-yl)aceticacid. An example synthesis of 1 is depicted in Scheme 8 and Example 1.

Example 1 Step 1

A round-bottom flask charged with [Ir(cod)C_(1]2) (350 mg, 0.52 mmol)and 1,4-bis(diphenylphosphanyl)butane (446 mg, 1.04 mmol) was flushedwith argon. DCM (60 mL), pinacolborane (3 mL, 21 mmol) andtert-butyl-3-(tert-butyldimethylsilyloxy)pent-4-enoate XXXVII [J. Org.Chem., (1994), 59(17), 4760-4764] (5 g, 17.48 mmol) in 5 mL of DCM wereadded successively at room temperature. The mixture was then stirred atroom temperature for 16 h. The reaction was quenched with MeOH (3 mL)and water (10 mL), the product was extracted with ether, and dried.Chromatography on silica gel (100% DCM→50% EtOAc/DCM gave tert-butyl3-(tert-butyldimethylsilyloxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pentanoateXXXVIII (5.5 g, 13.2 mmol, 75.5% yield).

Step 2

To a solution of tert-butyl3-(tert-butyldimethylsilyloxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pentanoateXXXVIII (5.4 g, 13 mmol) in THF (25 mL) was added(1S,2S,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptane-2,3-diol (2.4 g, 14.3mol) at room temperature. The reaction mixture was stirred for 16 h andthen was concentrated under vacuum. The residue was purified by columnchromatography (100% hexane→40% EtOAc/hexane) on silica gel to give1-(tert-butoxy)-3-[(tert-butyldimethylsilyl)oxy]-1-oxo-6-[(2S,6R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hexan-3-yl XXXIX (5.5 g, 11 mmol, 84.6%yield).

Step 3

To a solution of DCM (1.5 mL, 23.6 mmol) in THF (30 mL) at −100° C. wasadded 2.5 M n-butyl lithium in hexane (5.19 mL, 12.98 mmol) slowly undernitrogen and down the inside wall of the flask whilst maintaining thetemperature below −90° C. The resulting white precipitate was stirredfor 30 minutes before the addition of1-(tert-butoxy)-3-[(tert-butyldimethylsilyl)oxy]-1-oxo-6-[(2S,6R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hexan-3-yl XXXIX (5.5 g, 11 mmol) in THF (10mL) at −90° C. Zinc chloride (23.6 mL, 0.5 M in diethyl ether, 11.86mmol) was then added to the reaction mixture at −90° C. and then thereaction was allowed to warm to room temperature where it was stirredfor 16 h. The reaction was quenched with a saturated solution ofammonium chloride and the phases were separated. The aqueous phase wasthen extracted with diethyl ether (3×50 mL) and the combined organicextracts were dried over Na₂SO₄, filtered and concentrated under reducedpressure. The concentrated material was then chromatographed (100%hexane→50% EtOAc/hexane) to obtain6-(tert-butoxy)-4-[(tert-butyldimethylsilyl)oxy]-1-chloro-6-oxo-1-[(2S,6R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hexylXL (5.6 g, 10.5 mmol, 95.4% yield).

Step 4-5

Chloro intermediate XL (1.2 g, 2.33 mmol) in THF (10 mL) was cooled to−78° C. under nitrogen. A solution of LiHMDS (2.33 mL, 1.0 M in THF,2.33 mmol) was added slowly and the reaction flask was then allowed towarm to room temperature where it was stirred for 16 h. Method A: Theresulting was cooled to −78° C. and 5-thiopheneacetyl chloride was addedand the solution stirred at −78° C. for 1.5 h. Then, the cooling bathwas removed and the solution stirred at ambient temperature for 1.5 h.The reaction was quenched with water and extracted twice with EtOAc. Theorganic layers were combined, washed with water, brine, dried (Na₂SO₄)and concentrated in vacuo to afford a pale yellow solid as crudeproduct. The residue was chromatographed on a silica column (100%DCM→40% EtOAc/DCM) to afford 570 mg of6-(tert-butoxy)-4-[(tert-butyldimethylsilyl)oxy]-6-oxo-1-(thiophen-2-ylacetamido)-1-[(2S,6R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hexylidyneXLII as a white solid (570 mg, 0.92 mmol, 39.5% yield).

Step 6

Method D: To a solution of amide XLII (250 mg, 0.40 mmol) in 1,4-dioxane(10 mL) was added 10 mL of 3 N HCl. The mixture was heated to 110° C.for 90 min. The solution was cooled and diluted with 10 mL of water andextracted twice with 10 mL of diethyl ether. The aqueous layer wasconcentrated to afford a sticky residue as crude product. The residuewas rinsed with 5 mL of water, dissolved in 10% MeCN-water andlyophilized to afford2-((3R)-2-hydroxy-3-(2-(thiophen-2-yl)acetamido)-1,2-oxaborinan-6-yl)aceticacid 1 as white powder (100 mg, 0.337 mmol, 84.1% yield). ¹H NMR (CD₃OD)δ ppm 0.94-1.35 (m, 1H), 1.35-1.54 (m, 1H), 1.54-1.68 (m, 1H), 1.68-2.00(m, 1H), 2.20-2.67 (m, 3H), 3.93 (s, 1H), 3.98 (s, 1H), 4.02-4.23 (m,2H), 6.98-7.05 (m, 2H), 7.32-7.36 (m, 1H); ESIMS found for C₁₂H₁₆BNO₅Sm/z 280 (100%) (M−H₂O)+.

Alternative procedures for Steps 5 and 6 are shown in Scheme 9.

Step 5, Method B

To a solution of the acid (0.36 mmol) in DCM (10 mL) at 0° C. undernitrogen was added EDCI (86 mg, 0.45 mmol) and HOBT (48 mg, 0.36 mmol).After stirring at 0° C. for 30 minutes, a solution of the bis-silylamide intermediate XLI (0.3 mmol) in DCM (2 mL) followed byN-methyl-morpholine (65 μL, 0.6 mmol) were sequentially added at 0° C.The reaction flask was then allowed to warm to room temperature. Afterstirring at room temperature overnight, the reaction mixture was washedwith water, then brine, dried (Na₂SO₄), filtered and concentrated undervacuum. The residue was purified by column chromatography to produceintermediate XLIII.

Step 5, Method C

A solution of bis-silyl amide XLI (0.5 mmol) and acid in dry DCM (10 mL)were cooled to 0° C. Then DIPEA (1.5 mmol) was added drop wise followedHATU (0.75 mmol). The mixture was then allowed to warm to roomtemperature. After TLC has indicated complete conversion (˜3 h) of thestarting materials, the reaction was diluted with additional DCM (20mL). The reaction mixture was washed with water (3×5 mL), brine (10 mL),and dried over Na₂SO₄. After removal of the solvent, the residue wassubjected to flash column chromatography to produce intermediate XLIII.

Step 6, Method E

To a solution of amide (XLIII) (0.1 mmol) in dichloroethane (2 mL) at 0°C. was treated with pre-cooled 90% aq. TFA (4 mL) and stirred at roomtemperature for 3 hrs. The reaction mixture was evaporated in vacuo,azeotroped with MeCN (3×5 mL) and the residue was triturated with ether(5 mL). The product separated was filtered, dissolved in dioxane-watermixture and freeze dried to give the final product XLIV as a fluffysolid.

The following compounds are prepared in accordance with the proceduredescribed in the above Example 1 using methods A and D.

2-((3R)-2-hydroxy-3-(2-phenylacetamido)-1,2-oxaborinan-6-yl)acetic acid2. ¹H NMR (CD₃OD) δ ppm 0.82-1.33 (m, 1H), 1.33-1.51 (m, 1H), 1.51-1.68(m, 1H), 1.69-2.00 (m, 1H), 2.14-2.34 (m, 1H), 2.34-2.69 (m, 2H),3.74-3.76 (m, 2H), 3.98-4.20 (m, 1H), 7.22-7.41 (m, 5H); ESIMS found forC14H18BNO5 m/z 274 (100%) (M−H₂O)⁺.

2-((3R)-3-acetamido-2-hydroxy-1,2-oxaborinan-6-yl)acetic acid 3. ¹H NMR(CD₃OD) δ ppm 1.07-1.36 (m, 1H), 1.36-1.59 (m, 1H), 1.59-1.73 (m, 1H),1.73-2.09 (m, 1H), 2.15-2.16 (d, 3H), 2.35-2.69 (m, 3H), 4.01-4.23 (m,1H); ESIMS found for C₈H₁₄BNO₅ m/z 198 (100%) (M−H₂O)⁺.

2-((3R)-3-(cyclopropanecarboxamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 4. ¹H NMR (CD₃OD) δ ppm 0.98-1.32 (m, 5H), 1.32-1.67 (m, 2H),1.67-2.06 (m, 2H), 2.27-2.66 (m, 3H), 3.98-4.16 (m, 1H); ESIMS found forC₁₀H₁₆BNO₅ m/z 224 (100%) (M−H₂O)⁺.

The following compounds are prepared starting from enantiomerically pure(R)-tert-butyl 3-hydroxypent-4-enoate (J. Am. Chem. Soc. 2007, 129,4175-4177) in accordance with the procedure described in the aboveExample 1.

2-((3R,6S)-2-hydroxy-3-(2-(thiophen-2-yl)acetamido)-1,2-oxaborinan-6-yl)aceticacid 5. ¹H NMR (CD₃OD) δ ppm 0.97-1.11 (q, 1H), 1.47-1.69 (m, 2H),1.69-1.80 (m, 1H), 2.21-2.33 (td, 1H), 2.33-2.41 (dd, 1H), 2.58-2.67 (m,1H), 3.97 (s, 2H), 4.06-4.14 (m, 1H), 6.97-7.04 (m, 1H), 7.04-7.08 (m,1H), 7.34-7.38 (dd, 1H); ESIMS found for C₁₂H₁₆BNO₅S m/z 280 (100%)(M−H₂O)⁺.

2-((3R,6S)-2-hydroxy-3-(2-phenylacetamido)-1,2-oxaborinan-6-yl)aceticacid 6. ¹H NMR (CD₃OD) δ ppm 0.86-1.02 (m, 1H), 1.44-1.53 (dd, 1H),1.53-1.66 (td, 1H), 1.68-1.78 (m, 1H), 2.17-2.26 (dd, 1H), 2.26-2.36(dd, 2H), 3.75 (s, 2H), 4.02-4.12 (m, 1H), 7.22-7.40 (m, 5H); ESIMSfound for C₁₄H₁₈BNO₅ m/z 274 (100%) (M−H₂O)⁺.

The following compounds are prepared in accordance with the proceduredescribed in the above Example 1 starting from enantiomerically pure(R)-tert-butyl 3-hydroxypent-4-enoate (J. Am. Chem. Soc. 2007, 129,4175-4177) using methods B and D.

2-((3R,6S)-3-((S)-2-amino-2-phenylacetamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 33 was isolated as the HCl salt. ¹H NMR (CD₃OD) δ ppm 1.24-1.27 (m,1H), 1.51-1.72 (m, 3H), 2.45-2.50 (dd, J=5 Hz, J=5 Hz, 1H), 2.55-2.63(dd, J=2 Hz, J=3 Hz, 1H), 3.66-3.71 (m, 1H), 4.38-4.53 (m, 1H),4.99-5.09 (d, 1H), 7.48-7.56 (m, 5H); ESIMS found for C₁₄H₁₉BN₂O₅ m/z289 (M−H₂O)⁺.

2-((3R,6S)-3-(3-aminopropanamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 34 was isolated as the HCl salt. ¹H NMR (CD₃OD) δ ppm 1.24-1.29(td, J=13 Hz. J=3 Hz, 1H), 1.55-1.62 (td, J=14 Hz, J=4 Hz, 1H),1.68-1.72 (m, 1H), 1.79-1.82 (m, 1H), 2.43-2.47 (dd, J=6 Hz, J=6 Hz,2H), 2.70-2.74 (m, 2H), 2.83-2.86 (t, J=7 Hz, 2H), 3.26-3.29 (t, J=7 Hz,1H), 4.10-4.16 (m, 1H); ESIMS found for C₉H₁₇BN₂O₅ m/z 227 (M−H₂O)⁺.

(S)-2-amino-5-((3R,6S)-6-(carboxymethyl)-2-hydroxy-1,2-oxaborinan-3-ylamino)-5-oxopentanoicacid 35 was isolated as the HCl salt. ¹H NMR (CD₃OD) δ ppm 1.50-1.66 (m,2H), 1.66-1.84 (m, 2H), 2.10-2.20 (sex, J=8 Hz 1H), 2.20-2.29 (m, 1H),2.40-2.47 (m, 2H), 2.55-2.59 (q, J=7 Hz 1H), 2.69-2.75 (m, 1H),2.94-2.98 (td, J=9 Hz, J=2 Hz 1H), 3.99-4.12 (m, 2H); ESIMS found forC₁₁H₁₉BN₂O₇ m/z 302.8 (M+H).

2-((3R,6S)-3-(2-amino-4-(methylthio)butanamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 41 was isolated as the HCl salt. ¹H NMR (CD₃OD) δ ppm 1.45-1.65 (m,1H), 1.65-1.75 (m, 1H), 1.75-1.86 (m, 1H), 1.86-2.05 (m, 1H), 2.09-2.20(m, 4H), 2.46-2.73 (m, 6H), 2.84-2.86 (t, J=6 Hz, 1H), 3.99-4.02 (t, J=7Hz, 1H), 4.38-4.46 (m, 1H); ESIMS found for C₁₁H₂₁BN₂O₅S m/z 287(M−H₂O)⁺.

2-((3R,6S)-3-(2-(3,5-difluorophenyl)acetamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 66 was isolated as the HCl salt. ¹H NMR (CD₃OD) δ ppm 0.98-1.07 (q,J=13 Hz, 1H), 1.55-1.68 (m, 2H), 1.73-1.79 (dd, J=6 Hz, J=3 Hz, 1H),2.22-2.26 (dd, J=15 Hz, J=6 Hz, 1H), 2.33-2.38 (dd, J=13 Hz, J=7 Hz,1H), 2.62-2.63 (m, 1H), 3.78 (s, 2H), 4.05-4.12 (m, 1H), 6.88-5.93 (tt,J=5 Hz, J=2 Hz, 1H), 6.97-7.01 (dd, J=5 Hz, J=2 Hz, 2H); ESIMS found forC₁₄H₁₆BF₂NO₅ m/z 310.1 (M−H₂O)⁺.

The following compounds are prepared in accordance with the proceduredescribed in the above Example 1 starting from enantiomerically pure(R)-tert-butyl 3-hydroxypent-4-enoate (J. Am. Chem. Soc. 2007, 129,4175-4177) using methods A and E.

2-((3R,6S)-3-benzamido-2-hydroxy-1,2-oxaborinan-6-yl)acetic acid 37. ¹HNMR (CD₃OD) δ ppm 1.10-1.19 (q, J=11 Hz, 1H), 1.60-1.65 (dd, J=14 Hz,J=3 Hz, 1H), 1.71-1.80 (td, J=9 Hz, J=3 Hz, 1H), 1.91-1.96 (d, J=14 Hz,1H), 2.32-2.38 (dd, J=15 Hz, J=6 Hz, 1H), 2.44-2.49 (dd, J=15 Hz, J=7Hz, 1H), 2.82-2.84 (d, J=4 Hz, 1H), 4.10-4.17 (m, 1H), 7.57-7.60 (t, J=8Hz, 2H), 7.70-7.73 (t, J=8 Hz, 1H), 8.00-8.02 (d, J=8 Hz 2H); ESIMSfound for C₁₃H₁₆BNO₅ m/z 260 (M−H₂O)⁺.

The following compounds are prepared in accordance with the proceduredescribed in the above Example 1 starting from enantiomerically pure(R)-tert-butyl 3-hydroxypent-4-enoate (J. Am. Chem. Soc. 2007, 129,4175-4177) using methods B and E.

2-((Z)-1-(2-aminothiazol-4-yl)-2-((3R,6S)-6-(carboxymethyl)-2-hydroxy-1,2-oxaborinan-3-ylamino)-2-oxoethylideneaminooxy)-2-methylpropanoicacid 36 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.60 (s, 3H),1.61 (s, 3H), 1.62-1.75 (m, 2H), 1.77-1.82 (m, 1H), 1.86-1.91 (m, 1H),2.55-2.58 (t, J=6 Hz, 2H), 2.90-2.94 (t, J=6 Hz, 2H), 4.37-4.42 (m, 1H),7.11 (s, 1H); ESIMS found for C₁₅H₂₁BN₄O₈S m/z 411 (M−H₂O)⁺.

2-((3R,6S)-2-hydroxy-3-(3-phenylpropanamido)-1,2-oxaborinan-6-yl)aceticacid 38. ¹H NMR (CD₃OD) δ ppm 0.78-0.87 (q, J=13 Hz, 1H), 1.40-1.46 (dd,J=10 Hz, J=3 Hz, 1H), 1.54-1.62 (dt, J=8 Hz, J=4 Hz, 1H), 1.63-1.70 (d,J=13 Hz, 1H), 2.24-2.29 (dd, J=15 Hz, J=6 Hz, 1H), 2.36-2.40 (dd, J=8Hz, J=3 Hz, 1H), 2.53-2.56 (d, J=3.2 Hz, 1H), 2.74-2.78 (t, J=7 Hz, 2H),2.98-3.01 (t, J=6 Hz, 2H), 3.90-4.03 (m, 1H), 7.18-7.23 (m, 1H),7.25-7.33 (m, 4H); ESIMS found for C₁₅H₂₀BNO₅ m/z 288 (M−H₂O)⁺.

2-((3R,6S)-3-(2-(2-aminothiazol-4-yl)acetamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 39 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.25-1.36 (m,1H), 1.63-1.76 (m, 3H), 2.40-2.43 (d, J=6 Hz 2H), 2.68-2.70 (m, 1H),3.72 (s, 2H), 4.17-4.21 (m, 1H), 6.69 (s, 1H); ESIMS found forC₁₁H₁₆BN₃O₅S m/z 296.1 (M−H₂O)⁺.

2-((3R,6S)-3-((Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 40 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.56-1.67 (m,2H), 1.76-1.81 (m, 1H), 1.86-1.90 (m, 1H), 2.50-2.54 (dd, J=17 Hz, J=6Hz, 1H), 2.59-2.64 (dd, J=16 Hz, J=7 Hz, 1H), 2.86-2.90 (t, J=7 Hz, 1H),4.22 (s, 3H), 4.34-4.37 (m, 1H), 7.86 (s, 1H); ESIMS found forC₁₂H₁₇BN₄O₆S m/z 339.1 (M−H₂O)⁺.

2-((3R,6S)-3-(2-amino-3-(pyridin-3-yl)propanamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 42 was isolated as the TFA salt. ¹H NMR (CD₃OD/CF₃O₂D) δ ppm1.43-1.56 (m, 2H), 1.72-1.83 (m, 2H), 2.37-2.42 (m, 1H), 2.53-2.57 (t,J=6 Hz, 1H), 2.89-2.93 (t, J=7 Hz, 1H), 3.37-3.43 (m, 2H), 4.17-4.21 (t,J=7 Hz, 1H), 4.41-4.46 (m, 1H), 8.06-8.10 (dd, J=6 Hz, J=3 Hz, 1H),8.53-8.57 (t, J=17 Hz, 1H), 8.80-8.81 (brd, J=4 Hz, 1H), 8.84-8.87 (brd,J=6 Hz, 1H); ESIMS found for C₁₄H₂₀BN₃O=m/z 304.2 (M−H₂O)⁺.

2-((3R,6S)-2-hydroxy-3-(2-(pyridin-3-yl)acetamido)-1,2-oxaborinan-6-yl)aceticacid 43 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.15-1.20 (m,1H), 1.59-1.63 (m, 1H), 1.68-1.74 (m, 2H), 2.29-2.34 (dd, J=15 Hz, J=6Hz, 2H), 2.66-2.68 (m, 1H), 3.94 (s, 2H), 4.11-4.18 (m, 1H), 7.82-7.85(dd, J=8 Hz, J=6 Hz, 1H), 8.30-8.32 (d, J=8 Hz, 1H), 8.68-8.70 (brd, J=5Hz, 1H), 8.72-8.75 (brs, 1H); ESIMS found for C₁₃H₁₇BN₂O₅ m/z 275(M−H₂O)⁺.

2-((3R,6S)-2-hydroxy-3-((S)-piperidine-2-carboxamido)-1,2-oxaborinan-6-yl)aceticacid 45 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.44-1.51 (m,1H), 1.54-1.80 (m, 5H), 1.80-1.91 (m, 2H), 1.91-1.98 (brd, J=12 Hz, 1H),2.16-2.21 (dd, J=13 Hz, J=2 Hz, 1H), 2.49-2.57 (non, J=7 Hz, 2H),2.75-2.78 (t, J=6 Hz, 1H), 2.98-3.03 (dt, J=13 Hz, J=3 Hz, 1H),3.36-3.39 (d, J=13 Hz, 1H), 3.79-3.82 (dd, J=12 Hz, J=4 Hz, 1H),4.34-4.38 (m, 1H); ESIMS found for C₁₂H₂₁BN₂O₅ m/z 267 (M−H₂O)⁺.

2-((3R,6S)-2-hydroxy-3-((R)-1,2,3,4-tetrahydroisoquinoline-3-carboxamido)-1,2-oxaborinan-6-yl)aceticacid 46 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.43-1.51 (m,1H), 1.56-1.63 (m, 1H), 1.75-1.83 (m, 1H), 1.86-1.94 (m, 1H), 2.46-2.57(dq, J=16 Hz, J=6 Hz, 2H), 2.82-2.86 (t, J=7 Hz, 1H), 3.18-3.24 (dd,J=17 Hz, J=12 Hz, 1H), 3.36-3.41 (dd, J=17 Hz, J=5 Hz, 1H), 4.21-4.24(dd, J=18 Hz, J=13 Hz, 1H), 4.36-4.40 (m, 1H), 4.42 (s, 2H), 7.23-7.25(m, 1H), 7.27-7.33 (m, 3H); ESIMS found for C₁₆H₂₁BN₂O₅ m/z 315(M−H₂O)⁺.

Following method E while the compound is still in 90% aq.trifluoroacetic acid (10 mL), 10% Pd/C (50 mg) was added. The reactionmixture was stirred under hydrogen for 6 h, filtered through Celite andrinsed with dichloroethane (10 mL). The filtrate was concentrated undervacuum and azeotroped with dichloroethane (2×10 mL). Triturating withether resulted in a precipitate which was filtered and washed with ether(5 mL) and dried to give2-((3R,6S)-3-((R)-2-amino-5-guanidinopentanamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 47 as the TFA salt (50 mg) as an off-white solid. ¹H NMR (CD₃OD) δppm 1.39-1.46 (m, 1H), 1.52-1.58 (m, 1H), 1.66-1.77 (m, 2H), 1.77-1.84(m, 1H), 1.87-1.95 (m, 3H), 2.34-2.38 (dd, J=17 Hz, J=3 Hz, 1H),2.63-2.68 (dd, J=17 Hz, J=7 Hz, 1H), 2.94-2.97 (dd, J=10 Hz, J=6 Hz,1H), 3.20-3.24 (dt, J=7 Hz, J=2 Hz, 2H), 3.86-3.88 (t, J=6 Hz, 1H),4.27-4.31 (m, 1H); ESIMS found for C₁₂H₂₄BN₅O₅ m/z 312.2 (M−H₂O)⁺.

2-((3R,6S)-3-(2-(2-aminoethylthio)acetamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 48 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.38-1.46 (m,1H), 1.46-1.54 (m, 1H), 1.71-1.78 (m, 1H), 1.84-1.92 (m, 1H), 2.30-2.34(dd, J=16 Hz, J=4 Hz, 1H), 2.56-2.61 (dd, J=16 Hz, J=6 Hz, 1H),2.80-2.83 (t, J=6 Hz, 1H), 2.89-2.97 (non, J=7 Hz, 2H), 3.17-3.24 (non,J=5 Hz, 2H), 3.37 (s, 2H), 4.15-4.20 (m, 1H); ESIMS found forC₁₀H₁₉BN₂O₅S m/z 273 (M−H₂O)⁺.

2-((3R,6S)-2-hydroxy-3-(2-(pyridin-4-yl)acetamido)-1,2-oxaborinan-6-yl)aceticacid 49 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.17-1.27 (m,1H), 1.60-1.67 (m, 1H), 1.67-1.76 (m, 2H), 2.32-2.43 (m, 2H), 2.68-2.70(t, J=4 Hz, 2H), 3.22-3.26 (t, J=7 Hz, 1H), 4.15-4.21 (m, 1H), 7.94-7.96(d, J=7 Hz, 2H), 8.75-8.79 (d, J=6 Hz, 2H); ESIMS found for C₁₃H₁₇BN₂O₅m/z 275.1 (M−H₂O)⁺.

2-((3R,6S)-3-(2-(4-aminocyclohexyl)acetamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 50 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.15-1.25 (m,1H), 1.44-1.88 (m, 10H), 2.05-2.13 (m, 1H), 2.19-2.21 (d, J=8 Hz, 1H),2.30-2.36 (dd, J=6 Hz, 1H), 2.38-2.47 (m, 3H), 2.61-2.63 (brd, J=3 Hz,1H), 3.18-3.22 (t, J=7 Hz, 1H), 4.04-4.11 (m, 1H); ESIMS found forC₁₄H₂₅BN₂O₅ m/z 295.1 (M−H₂O)⁺.

2-((3R,6S)-3-(2-(1-aminocyclohexyl)acetamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 51 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.23-1.34 (m,1H), 1.34-1.48 (m, 1H), 1.48-1.86 (m, 12H), 2.40-2.50 (m, 2H), 2.65-2.83(m, 2H), 3.22-3.26 (t, J=7 Hz, 1H), 4.11-4.18 (m, 1H); ESIMS found forC₁₄H₂₅BN₂O₅ m/z 295 (M−H₂O)⁺.

2-((3R,6S)-2-hydroxy-3-(2-((R)-piperidin-2-yl)acetamido)-1,2-oxaborinan-6-yl)aceticacid 52 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.27-1.37 (m,1H), 1.49-1.80 (m, 7H), 1.86-2.00 (brdd, J=11 Hz, 3H), 2.44-2.46 (d, J=6Hz, 2H), 2.61-2.65 (m, 1H), 2.72-2.73 (d, J=6 Hz, 1H), 3.03-3.09 (t,J=13 Hz, 1H), 3.41-3.45 (d, J=13 Hz, 1H), 3.47-3.56 (m, 1H), 4.15-4.21(m, 1H); ESIMS found for C₁₃H₂₃BN₂O₅ m/z 281 (M−H₂O)⁺.

2-((3R,6S)-2-hydroxy-3-(2-((S)-piperidin-2-yl)acetamido)-1,2-oxaborinan-6-yl)aceticacid 53 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.26-1.35 (m,1H), 1.48-1.59 (m, 1H), 1.59-1.68 (m, 2H), 1.68-1.81 (m, 3H), 1.87-2.00(m, 3H), 2.45-2.47 (d, J=7 Hz, 2H), 2.65-2.67 (t, J=4 Hz, 1H), 2.74-2.76(t, J=6 Hz, 2H), 3.03-3.08 (dt, J=13 Hz, J=3 Hz, 1H), 3.42-3.46 (brd,J=13 Hz, 1H), 3.47-3.55 (m, 1H), 4.12-4.19 (m, 1H); ESIMS found forC₁₃H₂₃BN₂O₅ m/z 298.1 (M+H).

2-((3R,6S)-2-hydroxy-3-(2-(2-phenyl-1H-imidazol-1-yl)acetamido)-1,2-oxaborinan-6-yl)aceticacid 54 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.36-1.44 (m,1H), 1.44-1.54 (m, 1H), 1.66-1.80 (m, 2H), 2.15 (s, 1H), 2.48-2.51 (m,J=6 Hz, 1H), 2.72-2.75 (t, J=7 Hz, 1H), 4.33-4.39 (m, 1H), 4.94-5.05 (m,2H), 7.65-7.76 (m, 7H); ESIMS found for C₁₇H₂₀BN₃O₅ m/z 358.2 (M+H).

2-((3R,6S)-2-hydroxy-3-(3-(2-methyl-1H-benzo[d]imidazol-1-yl)propanamido)-1,2-oxaborinan-6-yl)acetic acid 55. ¹H NMR(CD₃OD) δ ppm 0.92-1.00 (m, 1H), 1.47-1.53 (m, 1H), 1.58-1.62 (m, 2H),2.31-2.33 (d, J=7 Hz, 2H), 2.50-2.52 (t, J=4 Hz, 1H), 2.97 (s, 3H),3.08-3.20 (m, 2H), 4.04-4.10 (m, 1H), 4.77-4.81 (t, J=6 Hz, 2H),7.61-7.68 (m, 2H), 7.75-7.78 (d, J=7 Hz, 1H), 7.93-7.95 (d, J=7 Hz, 1H);ESIMS found for C₁₇H₂₂BN₃O₅ m/z 342.2 (M−H₂O)⁺.

2-((3R,6S)-3-(4-((1H-tetrazol-1-yl)methyl)benzamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 56. ¹H NMR (CD₃OD) δ ppm 1.10-1.21 (m, 1H), 1.58-1.64 (m, 1H),1.70-1.79 (m, 1H), 1.89-1.96 (m, 1H), 2.31-2.36 (dd, J=15 Hz, J=6 Hz,1H), 2.41-2.47 (m, 1H), 2.80-2.83 (brd, J=4 Hz, 1H), 4.11-4.17 (m, 1H),5.83 (s, 2H), 7.53-7.55 (d, J=8 Hz, 2H), 8.02-8.05 (d, J=8 Hz, 2H), 9.30(s, 1H); ESIMS found for C₁₅H₁₈BN₅O₅ m/z 342.0 (M−H₂O)⁺.

2-((3R,6S)-2-hydroxy-3-(2-(pyridin-2-yl)acetamido)-1,2-oxaborinan-6-yl)aceticacid 57 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.21-1.32 (m,1H), 1.59-1.67 (m, 2H), 1.67-1.75 (m, 2H), 2.29-2.40 (m, 3H), 2.67-2.72(m, 1H), 4.14-4.21 (m, 1H), 7.62-7.66 (t, J=6 Hz, 1H), 7.70-7.73 (d, J=8Hz, 1H), 8.14-8.18 (t, J=8 Hz, 1H), 8.65-8.67 (d, J=5 Hz, 1H); ESIMSfound for C₁₃H₁₇BN₂O₅ m/z 275.1 (M−H₂O)⁺.

The following compounds are prepared in accordance with the proceduredescribed in the above Example 1 using methods C and E.

2-((3R,6S)-3-(1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 58 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.14-1.29 (m,3H), 1.39-1.44 (brd, J=7 Hz, 2H), 1.56-1.63 (dd, J=14 Hz, J=3 Hz, 1H),1.70-1.80 (m, 1H), 1.92-1.99 (d, J=14 Hz, 1H), 2.33-2.38 (dd, J=15 Hz,J=6 Hz, 1H), 2.43-2.48 (dd, J=15 Hz, J=7 Hz, 1H), 2.85-2.86 (d, J=3 Hz,1H), 3.46-3.52 (m, 4H), 3.59-3.64 (m, 4H), 3.73-3.79 (m, 1H), 4.08-4.15(m, 1H), 7.66-7.67 (d, J=7 Hz, 1H), 8.00-8.03 (d, J=13 Hz, 1H), 8.81 (s,1H); ESIMS found for C₂₃H₂₈BFN₄O₆ m/z 469.2 (M−H₂O)⁺.

2-[(3R,6S)-2-hydroxy-3-[(2S,3S,5R)-3-methyl-4,4,7-trioxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4λ⁶-thia-1-azabicyclo[3.2.0]heptane-2-amido]-1,2-oxaborinan-6-yl]acetic acid 59. ¹H NMR(CD₃OD) δ ppm 1.43 (s, 3H), 1.49-1.57 (m, 1H), 1.72-1.81 (m, 3H),2.51-2.56 dd, J=15 Hz, J=6 Hz, 1H), 2.62-2.67 (dd, J=15 Hz, J=8 Hz, 1H),2.80-2.84 (m, 1H), 3.41-3.44 (dd, J=17 Hz, J=2 Hz, 1H), 3.63-3.67 (dd,J=16 Hz, J=5 Hz, 1H), 4.37-4.44 (m, 1H), 4.61 (s, 1H), 4.90-4.94 (dd,J=5 Hz, J=2 Hz, 1H), 5.16-5.19 (d, J=15 Hz, 1H), 5.25-5.28 (d, J=15 Hz,1H), 7.77 (s, 1H), 8.07 (s, 1H); ESIMS found for C₁₆H₂₂BN₅O₈S m/z 438(M−H₂O)⁺.

2-((3R,6S)-2-hydroxy-3-(3-(5-phenyl-1,3,4-oxadiazol-2-yl)propanamido)-1,2-oxaborinan-6-yl)aceticacid 60. ¹H NMR (CD₃OD) δ ppm 1.10-1.21 (m, 1H), 1.50-1.58 (dd, J=14 Hz,J=3 Hz, 1H), 1.59-1.68 (dt, J=11 Hz, J=5 Hz, 1H), 1.74-1.81 (brd, J=13Hz, 1H), 2.22-2.26 (dd, J=15 Hz, J=6 Hz, 1H), 2.30-2.34 (dd, J=15 Hz,J=7 Hz, 1H), 2.63-2.64 (d, J=4 Hz, 1H), 3.01-3.12 (sex, J=7 Hz, 2H),3.33-3.43 (sex, J=7 Hz, 2H), 4.03-4.09 (m, 1H), 7.54-7.62 (m, 3H),8.03-8.05 (d, J=8 Hz, 2H); ESIMS found for C₁₇H₂₀BN₃O₆ m/z 356.1(M−H₂O)⁺.

2-((3R,6S)-3-(2-(2-aminopyridin-4-yl)acetamido)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 61 was isolated as the TFA salt. ¹H NMR (CD₃OD) δ ppm 1.58-1.66 (m,1H), 1.67-1.78 (m, 3H), 2.31-2.36 (dd, J=15 Hz, J=6 Hz, 1H), 2.39-2.44(dd, J=15 Hz, J=7 Hz, 1H), 2.65-2.68 (t, J=4 Hz, 1H), 4.12-4.19 (m, 1H),6.85-6.87 (d, J=7 Hz, 1H), 6.99 (s, 1H), 7.81-7.82 (d, J=7 Hz, 1H);ESIMS found for C₁₃H₁₈BN₃O₅ m/z 290.1 (M−H₂O)⁺.

Following method E, the reaction mixture was evaporated in vacuo,azeotroped with MeCN (3×5 mL) and the residue was triturated with ether(5 mL). The precipitate was filtered, dissolved in dioxane-water mixtureand freeze dried to get2-((3R)-3-((Z)-2-(2-aminothiazol-4-yl)-2-((1,5-dihydroxy-4-oxo-1,4-dihydropyridin-2-yl)methoxyimino)acetamido)-2-hydroxy-1,2-oxaborinan-6-yl)acetic acid 62 asthe TFA (25 mg) salt as a fluffy solid. ESIMS found for C₁₇H₂₀BN₅O₉S m/z464.0 (M−H₂O)⁺.

Synthesis of 2-((3R)-3-amino-2-hydroxy-1,2-oxaborinan-6-yl)acetic acidhydrochloride 7. An example synthesis of Z is depicted in Scheme 10 andExample 2.

Step 1

6-(tert-butoxy)-4-[(tert-butyldimethylsilyl)oxy]-1-chloro-6-oxo-1-[(2S,6R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hexaneXLI (515 mg, 0.97 mmol) in THF (5 mL) was cooled to −78° C. undernitrogen. A solution of LiHMDS (1 mL, 1.0 M in THF, 1 mmol, 1.0 eq) wasadded slowly and the reaction flask was then allowed to warm to roomtemperature where it was stirred for 16 h. The yellow solution wasconcentrated under reduced pressure to give an oil. After hexane (10 mL)was added to the oil, a precipitate formed. This was then filteredthrough Celite and the filtrate concentrated under reduced pressure togive1-[bis(trimethylsilyl)amino]-6-(tert-butoxy)-4-[(tert-butyldimethylsilyl)oxy]-6-oxo-1-[(2S,6R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hexylXLII.

Step 2

The procedure is identical to that found in Example 1 method D. Compound7 was isolated as a white powder (120 mg, 0.573 mmol, 59.1% yield). ¹HNMR (CD₃OD) δ ppm 1.43-1.66 (m, 1H), 1.66-1.79 (m, 1H), 1.79-1.97 (m,1H), 1.97-2.30 (m, 1H), 2.40-2.71 (m, 3H), 4.34-4.54 (m, 1H); ESIMSfound for C₆H₁₂BNO₄ m/z 174 (63%) (M+H).

Synthesis of2-((3R)-2-hydroxy-3-(2-(thiophen-2-yl)acetamido)-1,2-oxaborepan-7-yl)aceticacid 63. An example synthesis of 63 is depicted in Scheme 11 and Example3.

Example 3 Step 1

To a solution of tert-butyl 3-hydroxypent-4-enoate, XLVI (674 mg, 3.92mmol) in DCM (15 mL) was added diisopropylallylboronate XLV (2 g, 11.76mmol) via syringe. To the mixture was then added Grubbs' firstgeneration catalyst (260 mg, 0.31 mmol, 7.5 mol %) and the vessel waspurged with argon. The reaction was heated at 65° C. under nitrogen for18 h. The mixture was concentrated under vacuum and the residue waspurified by flash column chromatography (100% hexane→30% EtOAc/hexane)to afford tert-butyl2-(2-hydroxy-3,6-dihydro-2H-1,2-oxaborinin-6-yl)acetate XLVII (770 mg,3.63 mmol, 92.7% yield).

Step 2

To a solution of tert-butyl2-(2-hydroxy-3,6-dihydro-2H-1,2-oxaborinin-6-yl)acetate XLVII (670 mg,3.16 mmol) in EtOAc (45 mL) was added 10% Pd/C (135 mg). The vessel wasevacuated by applying vacuum and flushed with hydrogen gas. The reactionwas stirred under hydrogen for 2 h. The mixture was filtered through aCelite pad and which was washed with additional EtOAc (15 mL).Concentration of the filtrate gave pure tert-butyl2-(2-hydroxy-1,2-oxaborinan-6-yl)acetate XLVIII (641 mg, 3.00 mmol,94.8% yield).

Step 3

To a solution of tert-butyl 2-(2-hydroxy-1,2-oxaborinan-6-yl)acetateXLVIII (641 mg, 3.00 mmol) in THF (20 mL) was added(1S,2S,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptane-2,3-diol (509 mg, 3mol) at room temperature. The reaction mixture was stirred for 16 h andconcentrated under vacuum. The residue was purified by columnchromatography (100% hexane→40% EtOAc/hexane) on silica gel to givetert-butyl3-hydroxy-6-[(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hexanoateXLIX (790 mg, 2.16 mmol, 71.9% yield).

Step 4

To a solution of alcohol XLIX (790 mg, 2.16 mmol) in DMF (7.5 mL) wasadded imidazole (548 mg, 8.06 mmol) followed by TBDMSCl (580 mg, 3.87mol). The reaction mixture was stirred at room temperature for 16 h andconcentrated under vacuum. The white slurry was dissolved in 100 mL ofEtOAc and washed with saturated NaHCO₃ solution (20 mL), water (2×10 mL)and dried (Na₂SO₄). The organic extract was concentrated under vacuumand the residue was purified by column chromatography (100% hexane-30%EtOAc/hexane) on silica gel to give tert-butyl3-[(tert-butyldimethylsilyl)oxy]-6-[(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hexanoate L (1 g, 2.08 mmol, 96.3% yield).

Step 5

To a solution of DCM (0.26 mL, 4.16 mmol) in THF (5 mL) at −100° C. wasadded 2.5 M n-butyl lithium in hexane (1 mL, 2.5 mmol) slowly undernitrogen and down the inside wall of the flask whilst maintaining thetemperature below −90° C. The resulting white precipitate was stirredfor 30 minutes before the addition of L (1 g, 2.08 mmol) in THF (3 mL)at −90° C. Zinc chloride (5 mL, 0.5 M in THF, 2.5 mmol) was then addedto the reaction mixture at −90° C. and then the reaction was allowed towarm to room temperature where it was stirred for 16 h. The reaction wasquenched with a saturated solution of ammonium chloride and the phaseswere separated. The aqueous phase was then extracted with diethyl ether(2×10 mL) and the combined organic extracts were dried over Na₂SO₄,filtered and concentrated under reduced pressure. The concentratedmaterial was then chromatographed (100% hexane→20% EtOAc-hexane) toobtain tert-butyl(7S)-3-[(tert-butyldimethylsilyl)oxy]-7-chloro-7-[(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]heptanoateLI (740 mg, 1.40 mmol, 67.2% yield).

Step 6

Chloro intermediate LI (727 mg, 1.37 mmol) in THF (7 mL) was cooled to−78° C. under nitrogen. A solution of IM LiHMDS solution in THF (1.37mL, 1.37 mmol) was added slowly at −78° C. Upon completion of theaddition, the reaction flask was allowed to warm to room temperature.After stirring at room temperature for 16 h, the reaction mixture wasconcentrated under vacuum and hexane (20 mL) was added. The precipitatedlithium salts were filtered off through a Celite pad, rinsed withadditional hexane and the combined filtrates were concentrated undervacuum to give crude tert-butyl(7S)-7-[bis(trimethylsilyl)amino]-3-[(tert-butyldimethylsilyl)oxy]-7-[(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0²,6]decan-4-yl]heptanoateLII.

Step 7

To a stirred solution of 2-thiophenacetic acid (232 mg, 1.64 mmol) inDCM (45 mL) at 0° C. under nitrogen was added EDCI (391 mg, 2.05 mmol)and HOBT (221 mg, 1.64 mmol). After stirring at 0° C. for 30 minutes, asolution of the bis-silyl amide LII intermediate (1.37 mmol) in DCM (10mL) followed by N-methyl-morpholine (0.3 mL, 2.74 mmol) weresequentially added at 0° C. Upon completion of the addition, thereaction flask was allowed to warm to room temperature. After stirringat room temperature overnight, the reaction mixture was washed withwater, dried and concentrated under vacuum. The residue was purified bycolumn chromatography (100% DCM→50% EtOAc/DCM) to afford tert-butyl(7S)-3-[(tert-butyldimethylsilyl)oxy]-7-[2-(thiophen-2-yl)acetamido]-7-[(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]heptanoateLIII (340 mg, 0.54 mmol, 39.4% yield for 2 steps).

Step 8

To a solution of amide LIII (300 mg, 0.47 mmol) in 1,4-dioxane (9 mL)was added 9 mL of 3 N HCl. The reaction mixture was heated at reflux for90 minutes. The cooled reaction mixture was then diluted with water (10mL) and extracted with diethyl ether (2×10 mL). The aqueous layer wasconcentrated to afford a sticky solid which was azeotroped with MeCN(3×10 mL). The residue was dissolved in 40% dioxane-water andlyophilized to afford2-((3R)-2-hydroxy-3-(2-(thiophen-2-yl)acetamido)-1,2-oxaborepan-7-yl)aceticacid 63 as an off-white solid (100 mg, 32.1 mmol, 68.4% yield). ¹H NMR(CD₃OD) δ ppm 1.21-1.38 (m, 2H), 1.42-1.60 (m, 2H), 1.60-1.72 (m, 1H),1.80-1.94 (m, 1H), 2.32-2.47 (m, 2H), 2.54-2.58 (dd, J=15 Hz, J=6 Hz,1H), 3.97-3.98 (d, J=8 Hz, 1H), 4.05 (s, 2H), 6.97-7.01 (m, 1H),7.02-7.10 (m, 1H), 7.33-7.37 (m, 1H); ESIMS found for C₁₃H₁₈BNO₅S m/z294.0 (M−H₂O)⁺.

Synthesis of2-((3R)-2-hydroxy-3-(2-(thiophen-2-yl)acetamido)-2,3,4,7-tetrahydro-1,2-oxaborepin-7-yl)aceticacid 64. An example synthesis of 64 is depicted in Scheme 12 and Example4.

Example 4 Step 1

To a stirred solution of tert-butyl2-(2-hydroxy-3,6-dihydro-2H-1,2-oxaborinin-6-yl)acetate XLVII (770 mg,4.58 mmol) in THF (25 mL) was added(1S,2S,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptane-2,3-diol (980 mg,4.58 mmol) at room temperature. The reaction mixture was stirred for 16h and concentrated under vacuum. The residue was purified by columnchromatography (100% hexane-30% EtOAc/hexane) on silica gel to givetert-butyl(4Z)-3-hydroxy-6-[(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hex-4-enoateLIV (1 g, 2.75 mmol, 59.9% yield).

Step 2

To a solution of alcohol LIV (650 mg, 1.78 mmol) in DMF (10 mL) wasadded imidazole (484 mg, 7.12 mmol) followed by TBDMSCl (534 mg, 3.56mol). The reaction mixture was stirred at room temperature for 16 h andconcentrated under vacuum. The white slurry was dissolved in 100 mL ofEtOAc and washed with water (2×10 mL), brine and dried (Na₂SO₄). Theorganic extract was concentrated under vacuum and the residue waspurified by column chromatography (100% hexane→20% EtOAc/hexane) onsilica gel to give tert-butyl(4Z)-3-[(tert-butyldimethylsilyl)oxy]-6-[(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hex-4-enoateLV (800 mg, 1.67 mmol, 93.9% yield).

Step 3

To a solution of DCM (0.3 mL, 4.68 mmol) in THF (8 mL) at −100° C. wasadded 2.5 M n-butyl lithium in hexane (1.12 mL, 2.8 mmol) slowly undernitrogen and down the inside wall of the flask whilst maintaining thetemperature below −90° C. The resulting white precipitate was stirredfor 30 minutes before the addition of LV (1.12 g, 2.34 mmol) in THF (3mL) at −90° C. and the reaction was allowed to warm to room temperaturewhere it was stirred for 16 h. The reaction was quenched with asaturated solution of ammonium chloride and the phases were separated.The aqueous phase was then extracted with diethyl ether (2×10 mL) andthe combined organic extracts were dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The concentrated material was thenchromatographed (100% hexane→20% EtOAc/hexane) to obtain tert-butyl(4Z,7S)-3-[(tert-butyldimethylsilyl)oxy]-7-chloro-7-[(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hept-4-enoateLVI (820 mg, 1.56 mmol, 66.5% yield).

Step 4

Chloro intermediate LVI (790 mg, 1.49 mmol) in THF (10 mL) was cooled to−78° C. under nitrogen. A solution of IM LiHMDS solution in THF (1.5 mL,1.5 mmol) was added slowly at −78° C. Upon completion of the addition,the reaction flask was allowed to warm to room temperature. Afterstirring at room temperature for 16 h, the reaction mixture wasconcentrated under vacuum and hexane (20 mL) was added. The precipitatedlithium salts were filtered off through a Celite pad, rinsed withadditional hexane and the combined filtrates were concentrated undervacuum to give crude tert-butyl(4Z,7S)-7-[bis(trimethylsilyl)amino]-3-[(tert-butyldimethylsilyl)oxy]-7-[(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hept-4-enoateLVII.

Step 5

To a stirred solution of 2-thiophenacetic acid (252 mg, 1.78 mmol) inDCM (35 mL) at 0° C. under nitrogen was added EDCI (426 mg, 2.23 mmol)and HOBT (240 mg, 1.78 mmol). After stirring at 0° C. for 30 minutes, asolution of the crude bis-silyl amide LVII intermediate in DCM (10 mL)followed by N-methyl-morpholine (0.32 mL, 3 mmol) were sequentiallyadded at 0° C. Upon completion of the addition, the reaction flask wasallowed to warm to room temperature. After stirring at room temperatureovernight, the reaction mixture was washed with water, dried andconcentrated under vacuum. The residue was purified by columnchromatography (100% DCM→25% EtOAc/DCM) to afford tert-butyl(4Z,7S)-3-[(tert-butyldimethylsilyl)oxy]-7-[2-(thiophen-2-yl)acetamido]-7-[(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0^(2,6)]decan-4-yl]hept-4-enoateLVIII (600 mg, 0.95 mmol, 63.7% yield for 2 steps).

Step 6

A solution of amide LVIII (100 mg, 0.15 mmol) in anisole (5 mL) at 0° C.was treated with pre-cooled 90% aq trifluoroacetic acid (10 mL). Thereaction mixture was warmed to room temperature and stirred for 16 h.The mixture was evaporated in vacuo, azeotroped with MeCN (3×5 mL). Theresidue was sonicated in water (10 mL) and ether (10 mL). The aqueousphase was separated, washed with ether (2×5 mL) and freeze dried to givefluffy solid2-((3R)-2-hydroxy-3-(2-(thiophen-2-yl)acetamido)-2,3,4,7-tetrahydro-1,2-oxaborepin-7-yl)aceticacid 64 (15 mg, 0.05 mmol, 32.3% yield). ¹H NMR (CD₃OD) δ ppm 2.23-2.35(m, 2H), 2.40-2.61 (m, 2H), 2.76-2.83 (m, 1H), 3.96-4.03 (m, 1H), 4.10(s, 2H), 5.34-5.40 (m, 1H), 5.53-5.74 (m, 1H), 6.97-7.08 (m, 2H),7.32-7.39 (m, 1H); ESIMS found for C₁₃H₁₆BNO₅S m/z 292 (M−H₂O)⁺.

Synthesis of ethyl2-((3R,6S)-2-hydroxy-3-(2-(thiophen-2-yl)acetamido)-1,2-oxaborinan-6-yl)acetate65. An example synthesis of 65 is depicted in Scheme 13 and Example 5.

Example 5 Step 1

To a solution of 5 (400 mg, 1.35 mmol) in 4 mL of absolute ethanol wasadded anhydrous IM HCl in EtOAc (4 mL, 4 mmol). The reaction was stirredat room temperature for 16 h. The mixture was then concentrated andazeotroped with acetonitrile (3×10 mL) to give a sticky solid. Ether (10mL) was added to the azeotroped sticky solid and the resultingprecipitate was filtered. The filtered solid was rinsed with additionalether (5 mL) and dried to give ethyl2-((3R,6S)-2-hydroxy-3-(2-(thiophen-2-yl)acetamido)-1,2-oxaborinan-6-yl)acetate65 (300 mg, 0.92 mmol, 68.5% yield). ¹H NMR (CD₃OD) δ ppm 0.98-1.09 (q,J=14 Hz, 1H), 1.23-1.26 (t, J=7 Hz, 3H), 1.49-1.54 (dd, J=14 Hz, J=3 Hz,1H), 1.57-1.64 (dt, J=11 Hz, J=2 Hz, 1H), 1.72-1.78 (brd, J=14 Hz, 1H),2.24-2.28 (dd, J=15 Hz, J=6 Hz, 1H), 2.34-2.39 (dd, J=15 Hz, J=8 Hz,1H), 2.63 (brs, 1H), 3.99 (s, 2H), 4.07-4.13 (q, J=4 Hz, 3H), 6.99-7.01(t, J=4 Hz, 1H), 7.05-7.06 (d, J=3 Hz, 1H), 7.35-7.36 (dd, J=5 Hz, J=1.3Hz, 1H); ESIMS found for C₁₄H₂₀BNO₅S m/z 308.1 (M−H₂O)⁺.

Synthesis of2-((3R,7R)-2-hydroxy-3-(2-(thiophen-2-yl)acetamido)-2,3,4,7-tetrahydro-1,2-oxaborepin-7-yl)aceticacid 67. An example synthesis of 67 is depicted in Scheme 14 and Example6.

Example 6 Step 1

Prepared starting from enantiomerically pure (R)-tert-butyl3-hydroxypent-4-enoate [J. Am. Chem. Soc. (2007), 129, 4175-4177] inaccordance with the procedure described in the above Step 1 of Example3.

Steps 2-7

Prepared in accordance with the procedure described in the above Steps1-6 of Example 4.

White fluffy solid (23 mg, 0.074 mmol, 47% yield). ¹H NMR (CD₃OD) δ ppm2.29-2.31 (m, 1H), 2.40-2.68 (m, 4H), 4.10 (m, 2H), 4.74-4.82 (m, 1H),5.35-5.38 (m, 1H), 5.53-5.58 (m, 1H), 6.98-7.05 (m, 2H), 7.32-7.36 (m,1H); ESIMS found for C₁₃H₁₆BNO₅S m/z 292 (M−H₂O)⁺.

Synthesis of2-((3R,7S)-2-hydroxy-3-(2-(thiophen-2-yl)acetamido)-2,3,4,7-tetrahydro-1,2-oxaborepin-7-yl)aceticacid 68. An example synthesis of 68 is depicted in Scheme 15 and Example7.

Example 7 Step 1

Prepared starting from enantiomerically pure (S)-tert-butyl3-hydroxypent-4-enoate [J. Med. Chem., (2010), 53, 4654-4667] inaccordance with the procedure described in the above Step 1 of Example3.

Steps 2-7

Prepared in accordance with the procedure described in the above Steps1-6 of Example 4.

White fluffy solid (45 mg, 0.146 mmol, 39% yield). ¹H NMR (CD₃OD) δ ppm2.15-2.18 (m, 1H), 2.29-2.38 (m, 2H), 2.66-2.72 (m, 2H), 3.88-3.91 (m,1H) 4.00 (s, 2H), 5.24-5.27 (m, 1H), 5.57-5.63 (m, 1H), 6.87-6.96 (m,2H), 7.24-7.28 (m, 1H); ESIMS found for C₁₃H₁₆BNO₅S m/z 292 (M−H₂O)⁺.

Synthesis of2-((3R,6S)-3-(benzyloxycarbonylamino)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 69. An example synthesis of 69 is depicted in Scheme 16 and Example8.

Example 8 Step 1

A solution of bis-silyl amide XLI (0.2 mmol) in DCM (5 mL) was cooled to0° C. and benzyl chloroformate (0.056 mL, 0.4 mmol) was added. Then, thecooling bath was removed and the solution stirred at ambient temperaturefor 16 h. The reaction was quenched with water and extracted twice withEtOAc. The organic layers were combined, washed with water, brine, dried(Na₂SO₄) and concentrated in vacuo to afford a pale yellow oil as crudeproduct. The residue was chromatographed on a silica column (100%DCM→40% EtOAc/DCM) to afford carbamate LXIII (90 mg, 0.143 mmol, 71.5%yield).

Step 2

A solution of carbamate LXIII (70 mg, 0.11 mmol) in anisole (5 mL) at 0°C. was treated with pre-cooled 90% aq trifluoroacetic acid (10 mL). Thereaction mixture was warmed to room temperature and stirred for 16 h.The mixture was evaporated in vacuo, azeotroped with MeCN (3×5 mL). Theresidue was sonicated in water (10 mL) and ether (10 mL). The aqueousphase was separated, washed with ether (2×5 mL) and freeze dried to give2-((3R,6S)-3-(benzyloxycarbonylamino)-2-hydroxy-1,2-oxaborinan-6-yl)aceticacid 69 as a fluffy solid (10 mg, 0.033 mmol, 29.6% yield). ESIMS foundfor C₁₄H₁₈BNO₆S m/z 289.9 (M−H₂O)⁺.

The following compound is prepared in accordance with the proceduredescribed in the above Example 8.

2-((3R,6S)-2-hydroxy-3-(isobutoxycarbonylamino)-1,2-oxaborinan-6-yl)aceticacid 70 as a off-white solid (20 mg, 0.073 mmol, 27% yield). ¹H NMR(CD₃OD) δ ppm 0.95 (d, J=7 Hz, 6H), 1.62-1.67 (m, 1H), 1.70-1.75 (m,2H), 1.87-1.90 (m, 2H), 2.42-2.60 (m, 3H), 3.77-3.86 (m, 2H), 4.35-4.38(m, 1H); ESIMS found for C₁₁H₂₀BNO₆S m/z 256 (M−H₂O)⁺.

Synthesis of2-((3R,6S)-2-hydroxy-3-(phenylsulfonamido)-1,2-oxaborinan-6-yl)aceticacid 71. An example synthesis of 71 is depicted in Scheme 17 and Example9.

Example 9 Step 1-2

Prepared in accordance with the procedure described in the above Steps1-2 of Example 8.

Off-white solid (30 mg, 0.096 mmol, 43% yield). ¹H NMR (CD₃OD) δ ppm1.57-1.83 (series of m, 4H), 2.49-2.71 (series of m, 3H), 4.35-4.89 (m,1H), 7.51-7.59 (m, 3H), 7.85-7.89 (m, 2H); ESIMS found for C₁₂H₁₆BNO₆Sm/z 296.1 (M—H₂O)⁺.

Synthesis of2-((3R,6S)-2-hydroxy-3-(3-phenylureido)-1,2-oxaborinan-6-yl)acetic acid72. An example synthesis of 72 is depicted in Scheme 18 and Example 10.

Example 10 Step 1

To a solution of bis-silyl amide XLI (0.2 mmol) in DCM (5 mL) at 0° C.was added a solution of TFA in hexane (0.6 mmol). The reaction wasstirred at 0° C. for 20 min before adding phenyl isocyanate (0.04 mL,0.4 mmol) followed by N,N-diisopropylethylamine (0.18 mL, 1 mmol). Thecooling bath was then removed and the solution was stirred at ambienttemperature for 16 h. The reaction was quenched with water and extractedtwice with EtOAc. The organic layers were combined, washed with water,brine, dried (Na₂SO₄) and concentrated in vacuo to afford a pale yellowoil as crude product. The residue was chromatographed on a silica column(100% DCM→25% EtOAc/DCM) to afford the pure urea (50 mg, 0.081 mmol,40.7% yield).

Step 2

Deprotection was performed following the procedure described above instep 2 of example 8 to give2-((3R,6S)-2-hydroxy-3-(3-phenylureido)-1,2-oxaborinan-6-yl)acetic acid72 as a white solid (20 mg, 0.068 mmol, 86% yield). ¹H NMR (CD₃OD) δ ppm1.24-1.31 (m, 1H), 1.56-1.64 (m, 2H) 1.78-1.81 (m, 1H), 2.36-2.40 (dd,J=15 Hz, J=6 Hz, 1H), 2.46-2.58 (dd, J=13 Hz, J=7 Hz, 1H), 2.68-2.71 (m,1H), 4.07-4.12 (m, 1H), 7.15-7.18 (m, 1H), 7.34-7.37 (m, 4H); ESIMSfound for C₁₃H₁₇BN₂O₅ m/z 275.1 (M−H₂O)⁺.

Illustrative compounds of Formula (I) are shown in Table 1. Somestructures are shown with defined configurations at selectedstereocenters but the shown stereochemistries are not meant to belimiting and all possible stereoisomers of the shown structures are tobe considered encompassed herein. Compounds of any absolute and relativeconfigurations at the stereocenters as well as mixtures of enantiomersand diastereoisomers of any given structure are also encompassed herein.

TABLE 1 Example Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

Example 11

The potency and spectrum of β-lactamase inhibitors was determined byassessing their antibiotic potentiation activity.

The potentiation effect is observed by the reduction of the minimuminhibitory concentration of β-lactam antibiotics in the presence ofβ-lactamase inhibitors (BLIs). The activity of BLIs in combination withceftazidime or biapenem is assessed by the checkerboard assay(Antimicrobial Combinations. In Antibiotics in Laboratory Medicine, Ed.Victor Lorian, M. D., Fourth edition, 1996, pp 333-338) using brothmicrodilution method performed as recommended by the NCCLS (NationalCommittee for Clinical Laboratory Standards (NCCLS). 1997. Methods forDilution of Antimicrobial Susceptibility Tests for Bacteria That GrowAerobically—Fourth Edition; Approved Standard. NCCLS Document M7-A4, Vol17 No. 2). In this assay, multiple dilutions of two drugs, namely BLIand β-lactam (ceftazidime or biapenem), are being tested, alone and incombination, at concentrations equal to, above and below theirrespective minimal inhibitory concentrations (MICs). BLIs aresolubilized in 10% DMSO at 10 mg/mL. Stock solutions are furtherdiluted, according to the needs of a particular assay, in Mueller HintonBroth (MHB). Stock solution can be stored at −80° C.

The checkerboard (CB) assay is performed in microtiter plates.Ceftazidime or biapenem are diluted in the x axis, each columncontaining a single concentration of antibiotic. BLIs are diluted in they axis, each row containing an equal concentration of BLI. The result ofthese manipulations is that each well of the microtiter plate contains aunique combination of concentrations of the two agents. The assay isperformed in MHB with a final bacterial inoculum of 5×105 CFU/mL (froman early-log phase culture). Microtiter plates are incubated during 20 hat 35° C. and are read using a microtiter plate reader (MolecularDevices) at 650 nm as well as visual observation using a microtiterplate reading mirror. The MIC is defined as the lowest concentration ofantibiotics, within the combination, at which the visible growth of theorganism is completely inhibited. Activity of BLIs is reported at MPC8,or the minimal potentiation concentration to reduce the MIC ofantibiotic 8-fold.

Ceftazidime potentiation is studied in strains of various bacteria thatare resistant to ceftazidime due to expression of β-lactamasehydrolyzing enzymes. The panel of strains used in checkerboardexperiments contains β-lactamases that belong to all the known classesof these enzymes: A, B, C and D. Activity of Compound 1 is tested at themaximum concentration of 40 μg/mL. At this concentration it shows noinhibition of growth of any bacteria tested, however at concentration aslow as 0.6 μg/mL it reduced MICs to ceftazidime 8-fold in some bacteria(Table 2). Based on CB results, 1 has a broad-spectrum β-lactampotentiation activity against the strains expressing β-lactamases.Compound 1 was the most potent against the strains expressing KPCs andother class A enzymes (CTX-M-3), and some class C (MIR-1, CMY-2), andclass D (OXA-2) enzymes.

TABLE 2 Strain Organism Description PCR Class MPC8 KP1005 Klebsiellapneumoniae ESBL CTX-M-14 A Z KP1009 Klebsiella pneumoniae ESBL CTX-M-15A Y EC1008 Escherichia coli ESBL CTX-M-3 A X KP1004 Klebsiellapneumoniae Serine carbapenemase KPC-2 A X KP1008 Klebsiella pneumoniaeSerine carbapenemase KPC-2 A X EC1007 Escherichia coli Serinecarbapenemase KPC-3 A X KP1010 Klebsiella pneumoniae ESBL SHV-12 A YKP1012 Klebsiella pneumoniae ESBL SHV-18 A Y ec306 Escherichia coliFirst ESBL described SHV-2 A Y ec307 Escherichia coli Common SHV ESBLSHV-4 A Y ec308 Escherichia coli Common SHV ESBL SHV-5 A Y EC1009Escherichia coli ESBL TEM-10 A Z ec302 Escherichia coli Common ESBL inUS TEM-10 A Z EC1012 Escherichia coli ESBL TEM-12 A Y ec303 Escherichiacoli Common ESBL in US TEM-12 A Y EC1011 Escherichia coli ESBL TEM-26 AZ ec304 Escherichia coli Common ESBL in US TEM-26 A Z ec300 Escherichiacoli Common ESBL in France TEM-3 A Y ec301 Escherichia coli ESBL TEM-6 AZ CF1000 Citrobacter freundii Hyper AmpC expression C Y ECL1003Enterobacter cloacae Hyper AmpC expression C Z ec310 Escherichia coli E.cloacae-like Amp-C ACT-1 C X EC1004 Escherichia coli pAmpC CMY-2 C XEC1010 Escherichia coli pAmpC CMY-6 C Y EC1014 Escherichia coli pAmpCDHA-1 C Z EC1006 Escherichia coli pAmpC FOX-5 C Y EC1016 Escherichiacoli pAmpC FOX-5 C Z ec309 Escherichia coli E. cloacae-like Amp-C MIR-1C X KP1007 Klebsiella pneumoniae ESBL OXA-10, qnrB4 D Y KX1000Klebsiella oxytoca ESBL OXA-2 D X X = MPC8 of 2.5 μg/mL or less. Y =MPC8 of greater than 2.5 μg/mL to 10 μg/mL. Z = MPC8 of greater than 10μg/mL.

Next, ceftazidime potentiation activity of several cyclic boronic acidester derivatives was tested using a larger panel of strains expressingβ-lactamase hydrolyzing enzymes. Ceftazidime MICs were determined aloneand in the presence of fixed concentrations of various cyclic boronicacid ester derivatives. Most compounds were tested at 10 μg/mL. Cyclicboronic acid ester derivatives were capable of reducing ceftazidime MICs4 to >64-fold depending on β-lactamase (Table 3).

TABLE 3 Ceftazidime MIC (μg/mL) with or without cyclic boronic acidester derivative 3 at 10 4 at 10 5 at 10 6 at 10 7 at 10 Strain OrganismDescription PCR Class Alone μg/mL μg/mL μg/mL μg/mL μg/mL KP1005Klebsiella ESBL CTX- A Z Z Z Z Z Z pneumoniae M-14 KP1009 KlebsiellaESBL CTX- A Z Z Z Z Z Z pneumoniae M-15 KP1006 Klebsiella ESBL CTX- A YX X X X ND pneumoniae M2 EC1008 Escherichia ESBL CTX- A Z Y Y Y Y Z coliM3 pa1063 Pseudomonas ESBL GES-1 A Z Z Z Z Z Z aeruginosa KP1004Klebsiella Serine KPC-2 A Z Y Y Y Y Z pneumoniae carbapenemase KP1008Klebsiella Serine KPC-2 A Y X X X X Z pneumoniae carbapenemase EC1007Escherichia Serine KPC-3 A Z X X X X Z coli carbapenemase KP1010Klebsiella ESBL SHV-12 A Z Z Z Y Y Z pneumoniae KP1012 Klebsiella ESBLSHV-18 A Z Z Z Y Y Z pneumoniae ec306 Escherichia First ESBL SHV-2 A Z ZZ Z Z Z coli described ec307 Escherichia Common SHV SHV-4 A Z Z Z Y Z Zcoli ESBL ec308 Escherichia Common SHV SHV-5 A Z Z Z Z Z Z coli ESBLKP1011 Klebsiella ESBL SHV-5 A Y X X X X ND pneumoniae EC1009Escherichia ESBL TEM-10 A Z Z Z Z Z Z coli ec302 Escherichia Common ESBLTEM-10 A Z Z Z Z Z Z coli in US EC1012 Escherichia ESBL TEM-12 A Z Z Z YY Z coli ec303 Escherichia Common ESBL TEM-12 A Z Z Z Y Y Z coli in USEC1011 Escherichia ESBL TEM-26 A Z Z Z Z Z Z coli ec300 EscherichiaCommon ESBL TEM-3 A Z Z Z Y Z Z coli in France ec301 Escherichia ESBLTEM-6 A Z Z Z Z Z Z coli KP1014 Klebsiella Metallo β- Vim-1 B Z Z Z Z ZND pneumoniae lactamase CF1000 Citrobacter Hyper AmpC C Z Z Z Y Y Zfreundii expression CF1001 Citrobacter Hyper AmpC C Z Z Z Y Y NDfreundii expression ECL1002 Enterobacter Hyper AmpC C Z Z Y Y Y Zcloacae expression ECL1003 Enterobacter Hyper AmpC C Z Z Z Z Z NDcloacae expression ec310 Escherichia E. cloacae -like ACT-1 C Z Y Y X XZ coli Amp-C EC1004 Escherichia pAmpC CMY-2 C Z Y Y Y Y Z coli SA1000Salmonella pAmpC CMY-2 C Z Z Y Y X ND KP1013 Klebsiella pAmpC CMY-2 C ZZ Y Y Y ND pneumoniae EC1010 Escherichia pAmpC CMY-6 C Z Z Y Y Y Z coliEC1014 Escherichia pAmpC DHA-1 C Z Y Y X X Z coli EC1006 EscherichiapAmpC FOX-5 C Z Y Z Y Y Z coli EC1016 Escherichia pAmpC FOX-5 C Z Z Z ZZ ND coli ec309 Escherichia E. cloacae -like MIR-1 C Z Y Y X X Z coliAmp-C PAM2005 Pseudomonas ampC C Z Z Z Z Z Z aeruginosa PAM2035Pseudomonas ampC mexA:tet C Z Z Z Y Y Z aeruginosa KP1007 KlebsiellaESBL OXA-10 D Z Z Z Y Y Z pneumoniae KX1000 Klebsiella ESBL OXA-2 D Z ZY Y Y Y oxytoca AB1054 Acinetobacter OXA- OXA-23 D Z Z Z Z Z Z baumanniicarbapenemase AB1052 Acinetobacter OXA- OXA-24 D Z Z Z Z Z ND baumanniicarbapenemase AB1057 Acinetobacter OXA- OXA-58 D Z Z Z Z Z Z baumanniicarbapenemase X = MIC of 1 μg/mL or less. Y = MIC of greater than 1μg/mL to 8 μg/mL. Z = MIC of greater than 8 μg/mL. ND = Not Determined.

Biapenem is a carbapenem β-lactam; only selected β-lactamases conferresistance to this class of antibiotics. Among them are serinecarbapemenases that belong to class A and class D. Biapenem potentiationis studied in strains expressing various carbapenemases from theseclasses using CB assays. Various cyclic boronic acid ester derivativesshowed significant potentiation of biapenem against the strainsexpressing class A carbapenemases: MPC8 (minimal potentiationconcentration of cyclic boronic acid ester derivative (μg/mL) to reducethe MIC of Biapenem 8-fold) varied from 0.02 μg/mL to 0.16 μg/mL (Table4). Cyclic boronic acid ester derivatives were capable of reducingbiapenem MICs up to 1000-fold (Table 4).

TABLE 4 Strain Organism Description PCR Class Compound MPC8 ECL1004Enterobacter cloacae Serine carbapenemase NMC-A A 1 Y EC1007 Escherichiacoli Serine carbapenemase KPC-3 A 1 X KP1004 Klebsiella pneumoniaeSerine carbapenemase KPC-2 A 1 Y SM1000 Serratia marcescens Serinecarbapenemase SME-2 A 1 Y ECL1004 Enterobacter cloacae Serinecarbapenemase NMC-A A 2 Y EC1007 Escherichia coli Serine carbapenemaseKPC-3 A 2 X KP1004 Klebsiella pneumoniae Serine carbapenemase KPC-2 A 2X SM1000 Serratia marcescens Serine carbapenemase SME-2 A 2 Y ECL1004Enterobacter cloacae Serine carbapenemase NMC-A A 3 X EC1007 Escherichiacoli Serine carbapenemase KPC-3 A 3 X KP1004 Klebsiella pneumoniaeSerine carbapenemase KPC-2 A 3 X KP1008 Klebsiella pneumoniae Serinecarbapenemase KPC-2 A 3 X SM1000 Serratia marcescens Serinecarbapenemase SME-2 A 3 Y AB1052 Acinetobacter baumanniiOXA-carbapenemase OXA-24 D 3 Z AB1054 Acinetobacter baumanniiOXA-carbapenemase OXA-23 D 3 Z AB1057 Acinetobacter baumanniiOXA-carbapenemase OXA-58 D 3 Z ECL1004 Enterobacter cloacae Serinecarbapenemase NMC-A A 4 X EC1007 Escherichia coli Serine carbapenemaseKPC-3 A 4 X KP1004 Klebsiella pneumoniae Serine carbapenemase KPC-2 A 4X KP1008 Klebsiella pneumoniae Serine carbapenemase KPC-2 A 4 X SM1000Serratia marcescens Serine carbapenemase SME-2 A 4 X AB1052Acinetobacter baumannii OXA-carbapenemase OXA-24 D 4 Z AB1054Acinetobacter baumannii OXA-carbapenemase OXA-23 D 4 Z AB1057Acinetobacter baumannii OXA-carbapenemase OXA-58 D 4 Z ECL1004Enterobacter cloacae Serine carbapenemase NMC-A A 5 Y EC1007 Escherichiacoli Serine carbapenemase KPC-3 A 5 X KP1004 Klebsiella pneumoniaeSerine carbapenemase KPC-2 A 5 X SM1000 Serratia marcescens Serinecarbapenemase SME-2 A 5 Y AB1052 Acinetobacter baumanniiOXA-carbapenemase OXA-24 D 5 Z AB1054 Acinetobacter baumanniiOXA-carbapenemase OXA-23 D 5 Z AB1057 Acinetobacter baumanniiOXA-carbapenemase OXA-58 D 5 Z ECL1004 Enterobacter cloacae Serinecarbapenemase NMC-A A 6 Y EC1007 Escherichia coli Serine carbapenemaseKPC-3 A 6 X KP1004 Klebsiella pneumoniae Serine carbapenemase KPC-2 A 6X SM1000 Serratia marcescens Serine carbapenemase SME-2 A 6 Y AB1052Acinetobacter baumannii OXA-carbapenemase OXA-24 D 6 Z AB1054Acinetobacter baumannii OXA-carbapenemase OXA-23 D 6 X AB1057Acinetobacter baumannii OXA-carbapenemase OXA-58 D 6 Z X = MPC8 of lessthan 0.16 μg/mL. Y = MPC8 of 0.16 μg/mL to 1 μg/mL. Z = MPC8 of greaterthan 1 μg/mL.

Example 12

The ability of β-lactamase inhibitors to inhibit hydrolysis ofceftazidime and biapenem was studied. Lysates were prepared frombacteria expressing various β-lactamases as a source of enzymes.Bacterial lysates were prepared as follows. A single colony from thefresh over-night plate was transferred to 5 mL of LB broth and grown toOD₆₀₀=0.6-0.8. Next, this culture was transferred to 500 mL of LB andgrown to OD₆₀₀=0.7-0.9. Cells were pelleted by centrifugation at 5000RPM (JA-14 rotor) for 15 minutes at room temperature. The pellet wasresuspended in 10 mL of PBS. Five freeze-thaw cycles by putting cells at−20° C. and thawing them at the room temperature were next applied.After the last thaw step cells were spun down at 18K for 30 minutes andthe supernatant was collected. This lysate was stored at −20° C.

Next, the activity of bacterial lysates was optimized for ceftazidimeand biapenem cleavage as follows. 50 al of buffer A (50 mM SodiumPhosphate pH=7; 0.5% glucose, 1 mM MgCl₂) was added to each well of96-well UV-transparent plate. 50 al of lysate was titrated vertically in96-well plate column to generate 2-fold lysate dilutions. 100 al ofbuffer A was added to each well, placed in plate reader at 37° C. andincubated for 15 minutes. 50 al of 50 μg/mL solutions of ceftazidime orbiapenem in buffer A (pre-incubated at 37° C. for 15 minutes) were addedto each well. Hydrolysis of ceftazidime and biapenem was measured at 250nm and 296 nm, respectively. This experiment was used to determine theoptimal lysate dilution which produced a linear curve of relative UVsignal that decreased to approximately OD=0.3-0.5 over 1 hour.

Finally, the potency of cyclic boronic acid ester derivative to inhibitthe cleavage of ceftazidime and biapenem cleavage by bacterial lysateswas determined. 100 al of buffer A (50 mM Sodium Phosphate pH=7; 0.5%glucose, 1 mM MgCl₂) was added to each well of 96-well UV-transparentplate. 50 μl of 6×cyclic boronic acid ester derivative solution inbuffer A was titrated vertically in 96-well plate column to generate3-fold dilutions. 50 μl of diluted lysate in buffer A (optimal dilutionis determined in experiment above) was added, and the plate wasincubated in the plate reader at 37° C. for 15 minutes. 50 μl of 50μg/mL solutions of ceftazidime or biapenem in buffer A (pre-incubated at37° C. for 15 minutes) were next added to each well and hydrolysis ofceftazidime or biapenem was recorded at 250 nm and 296 nm, respectively.EC₅₀ of inhibition was determined by plotting the rate of ceftazidime orbiapenem cleavage vs. cyclic boronic acid ester derivativeconcentration.

The results of these experiments are presented in Table 5 and Table 6.These experiments demonstrate that the described compounds areinhibitors with a broad-spectrum activity towards various β-lactamases.

TABLE 5 IC₅₀ (μg/mL) of inhibition of Ceftazidime hydrolysis StrainOrganism Description PCR Class Tazobactam 3 4 5 6 7 KP1005 KlebsiellaESBL CTX-M-14 A X Z Z X Y Z pneumoniae KP1009 Klebsiella ESBL CTX-M-15 AX Z Z X Y Y pneumoniae pa 1063 Pseudomonas ESBL GES-1 A Y Z Y X Y Yaeruginosa KP1004 Klebsiella Serine KPC-2 A Z X X X X Z pneumoniaecarbapenemase KP1008 Klebsiella Serine KPC-2 A Z Y X X X Z pneumoniaecarbapenemase EC1007 Escherichia coli Serine KPC-3 A Z Y X X X Zcarbapenemase KP1010 Klebsiella ESBL SHV-12 A X Z Z Y Z Z pneumoniaeKP1012 Klebsiella ESBL SHV-18 A X Z Z Y Y Z pneumoniae ec306 Escherichiacoli First ESBL SHV-2 A Y Z Z Y Y Z described ec308 Escherichia coliCommon SHV SHV-5 A X Z Z Z Z Z ESBL ec302 Escherichia coli Common ESBLin TEM-10 A X Y Z X Y Y US ec303 Escherichia coli Common ESBL in TEM-12A X Z Z Y Z Y US ec304 Escherichia coli Common ESBL in TEM-26 A X Z Z YY Y US ec300 Escherichia coli Common ESBL in TEM-3 A X Z Z Y Z Z Franceec301 Escherichia coli ESBL TEM-6 A X Z Z Y Y Y ECL1003 EnterobacterHyper AmpC C ND Z Z Y Z Z cloacae expression EC1014 Escherichia colipAmpC DHA-1 C ND Z Z Y Z Z KP1007 Klebsiella ESBL OXA-10 D Y Z Z Y Z Zpneumoniae KX1000 Klebsiella oxytoca ESBL OXA-2 D X Z Z X Y Z X = IC₅₀of less than 0.1 μg/mL. Y = IC₅₀ of 0.1 μg/mL to 1 μg/mL. Z = IC₅₀ ofgreater than 1 μg/mL. ND = Not Determined.

TABLE 6 IC₅₀ (μg/mL) of inhibition of biapenem hydrolysis StrainOrganism Description PCR Class Tazobactam 3 4 5 6 7 EC1007 Escherichiacoli Serine KPC-3 A Z Y Y X X Z carbapenemase KP1004 Klebsiella SerineKPC-2 A Z Z Y X Y ND pneumoniae carbapenemase KP1008 Klebsiella SerineKPC-2 A Z Z Z Y Y ND pneumoniae carbapenemase SM1000 Serratia SerineSME-2 A Y Z Y X Y Z marcescens carbapenemase X = IC₅₀ of less than 0.1μg/mL. Y = IC₅₀ of 0.1 μg/mL to 1 μg/mL. Z = IC₅₀ of greater than 1μg/mL. ND = Not Determined.

The potency and spectrum of β-lactamase inhibitors is also determined byassessing their aztreonam potentiation activity in a dose titrationpotentiation assay using strains of various bacteria that are resistantto aztreonam due to expression of various β-lactamases. Aztreonam is amonobactam antibiotic and, similar to ceftazidime, is hydrolyzed by themajority of beta-lactamases that belong to class A, C or D (but notclass B). The potentiation effect is observed as the ability of BLIcompounds to inhibit growth in the presence of sub-inhibitoryconcentration of aztreonam. MIC of test strains vary from 32 g/mLto >128 μg/mL. Aztreonam is present in the test medium at 4 μg/mL.Compounds were tested at the highest concentration of 40 μg/mL. In thisassay potency of compounds is determined as a concentration of BLIs toinhibit growth of bacteria in the presence of 4 g/mL of aztreonam(MPC_(@4)). Tables 7, 8 and 9 summarize BLI potency of aztreonampotentiation (MPC_(@4)) for various strains overexpressing class A(ESBLs), class A (KPCs), and class C and class D beta-lactamases,respectively. Aztreonam MIC for each strain is also shown. Table 7summarizes activity of BLIs to potentiate aztreonam against strainsexpressing class A ESBLs. Table 8 summarizes activity of BLIs topotentiate aztreonam against strains expressing class A KPCs. Table 9summarizes activity of BLIs to potentiate aztreonam against strainsexpressing class C and D enzymes.

TABLE 7 Aztreonam MIC (μg/mL) >128 >128 64 >128 32 128 >128 64 AZT AZTAZT AZT AZT AZT AZT AZT MPC₄ MPC₄ MPC₄ MPC₄ MPC₄ MPC₄ MPC₄ MPC₄ CTX-M-14CTX-M-15 SHV-5 SHV-12 SHV-18 TEM-10 TEM-10 TEM-26 KP1005 KP1009 ec308KP1010 KP1012 EC1009 ec302 ec304 Clavulanic Acid 1.25 1.25 0.08 0.040.04 0.16 0.3 0.04 Tazobactam 10 20 10 1.25 1.15 2.5 5 0.6 3 Z Z Z Z Z ZZ Z 4 Z Z Z Z Z Z Z Z 5 Z Y Y Y X Y Z Y 6 Z Z Z Y Y Y Z Y 7 Z Z Z Z Y XY Y 33 Z Z Z Z Z Z Z Z 34 Z Z Z Z Z Z Z Z 35 Z Z Z Z Z Z Z Z 36 Z Z Z YY Z Z Y 37 Z Z Z Y X Y Z Y 38 Z Z Z Y Z Z Z Z 39 Z Z Z Z Y Z Z Y 40 Z ZZ Y Z Z Z Z 41 Z Z Z Z Z Z Z Z 42 Z Z Z Z Z Z Z Z 43 Z Z Z Y Y Y Z Y 45Z Z Z Z Z Z Z Z 46 Z Z Z Z Z Z Z Z 47 Z Z Z Z Z Z Z Z 48 Z Z Z Z Z Z Z Z49 Z Z Z Z Y Z Z Z 50 Z Z Z Z Z Z Z Z 51 Z Z Z Z Y Y Z Z 52 Z Z Z Z Z ZZ Z 53 Z Z Z Z Z Z Z Z 54 Z Z Z Z Z Z Z Z 55 Z Z Z Z Z Z Z Y 56 Z Z Z ZZ Z Z Z 57 Z Z Z Z Y Z Z Z 58 Z Z Z Z Z Z Z Z 59 Z Z Z Z Z Z Z Z 60 Z ZZ Z Z Z Z Z 61 Z Z Z Y Y Z Z Z 62 X X X X X X Y X 63 Y Y Y X X Y Z Y 64Y Y X X X Y Y X 65 Z Z Z Z Z Z Z Z 66 Z Z Z Z Z Z Z Z X = MPC_(@4) ofless than 5 μg/mL. Y = MPC_(@4) of 5 μg/mL to 20 μg/mL. Z = MPC_(@4) ofgreater than 20 μg/mL. ND = Not Determined.

TABLE 8 Aztreonam MIC >128 64 >128 AZT AZT AZT MPC₄ MPC₄ MPC₄ KPC-2KPC-2 KPC-3 KP1004 KP1008 EC1007 Clavulanic Acid >40 20 40Tazobactam >40 >40 >40 3 X X X 4 X X X 5 X X X 6 X X X 33 X X X 34 X X X35 Y X X 36 Z Z Z 37 X X X 38 Z X X 39 Y X X 40 Z Y Z 41 Y X X 42 Y X X43 X X X 45 Z Y X 46 X X X 47 Z Y Y 48 X X X 49 X X X 50 X X X 51 X X X52 Y X X 53 Y X X 54 Z X Y 55 Y X X 56 Y X X 57 X X X 58 Z Z Z 59 Z Z Z60 Z Y Y 61 X X X 62 Y X Y 63 Z Y Y 64 Z X Y 65 Z Z Z 66 Y X X X =MPC_(@4) of less than 5 μg/mL. Y = MPC_(@4) of 5 μg/mL to 20 μg/mL. Z =MPC_(@4) of greater than 20 μg/mL. ND = Not Determined.

TABLE 9 Class C C C D D Aztreonam MIC 128 >128 AZT 128 64 AZT 32MPC_(@4) AZT AZT MPC_(@4) AZT OXA-10, MPC_(@4) MPC_(@4) CMY-6 MPC_(@4)qnrB4 OXA-2, ECL1002 EC1010 PAM2035 KP1007 KPX1001 Clavulanic >40 40 >400.08 5 Acid Tazobactam >40 20 20 5 >40 3 Z Z Z Z Z 4 Y Y Z Z Z 5 Y Y X XY 6 Y Z Y Y Z 33 Z Z Z Z Z 34 Z Z Z Z Z 35 Z Z Z Z Z 36 Z Z Z Y Z 37 Z ZZ Z X 38 Z Z Z Z Z 39 Z Z Z Z Z 40 Z Z Z Z Z 41 Z Z Z Z Z 42 Z Z Z Z Z43 Y Y Y Z Y 45 Z Z Z Z Z 46 Z Z Z Z Z 47 Z Z Z Z Z 48 Z Z Z Z Z 49 Z ZY Z Y 50 Z Z Z Z Y 51 Z Z Z Z Y 52 Z Z Z Z Z 53 Z Z Z Z Y 54 Z Z Z Z Z55 Z Z Z Z Z 56 Z Z Z Z Y 57 Z Z Z Z Z 58 Z Z Z Z Z 59 Z Z Z Z Z 60 Z ZZ Z Z 61 Y Y Y Y Y 62 Z X X X Y 63 Y Y Y Y Y 64 Y Z Y X Y 65 Z Z Z Z Z66 Z Z Z Z Z X = MPC_(@4) of less than 5 μg/mL. Y = MPC_(@4) of 5 μg/mLto 20 μg/mL. Z = MPC_(@4) of greater than 20 μg/mL. ND = Not Determined.

The potency and spectrum of β-lactamase inhibitors is also determined byassessing their biapenem potentiation activity in a dose titrationpotentiation assay using strains expressing serine carbapemenases (suchas KPC). The potentiation effect is observed as the ability of BLIcompounds to inhibit growth in the presence of sub-inhibitoryconcentration of biapenem. MIC of test strains vary from 4 μg/mL to >1μg/mL. Biapenem is present in the test medium at 1 μg/mL. Compoundstested at the highest concentration of 40 μg/mL. In this assay potencyof compounds is determined as a concentration of BLIs to inhibit growthof bacteria in the presence of 1 μg/mL of biapenem (MPC_(@1)). Table 10summarizes BLI potency of biapenem potentiation (MPC_(@1)). Biapenem MICfor each strain is also shown.

TABLE 10 Biapenem MIC >8 8 4 8 BPM BPM BPM BPM MPC_(@1) MPC_(@1)MPC_(@1) MPC_(@1) KP1004 KP1008 EC1007 ECL1004 KPC-2 KPC-2 KPC-3 NMC-ATazobactam 40 0.3 5 0.6 3 X X X Y 4 X X X X 5 X X X X 6 X X X X 33 X X XX 34 X X X Y 35 X X X Y 36 Z X Y X 37 X X X X 38 X X X X 39 X X X X 40 YX Y Y 41 X X X Y 42 X X X Y 43 X X X X 45 Y X X Z 46 X X X X 47 Y X X Z48 X X X X 49 X X X X 50 X X X X 51 X X X Y 52 X X X Y 53 X X X Y 54 X XX X 55 X X X X 56 X X X X 57 X X X X 58 Z Z Z Z 59 Y X X X 60 X X X X 61X X X X 62 X X X X 63 Y X Y Y 64 Y X X X 65 Y X Y Z 66 X X X X X =MPC_(@1) of less than 1 μg/mL. Y = MPC_(@1) of 1 μg/mL to 5 μg/mL. Z =MPC_(@1) of greater than 5 μg/mL. ND = Not Determined.

Some bacterial lysates were also optimized for the cleavage of aztreonamand nitrocefin. EC₅₀ of inhibition was determined by plotting the rateof aztreonam or nitrocefin cleavage vs. BLI concentration. The resultsof these experiments are presented in Table 11. These experimentsconfirmed that the described compounds are inhibitors with abroad-spectrum activity towards various β-lactamases.

TABLE 11 NCF IC₅₀ AZT IC₅₀ AZT IC₅₀ AZT IC₅₀ AZT IC₅₀ AZT IC₅₀ AZT IC₅₀AZT IC₅₀ AZT IC₅₀ AZT IC₅₀ EC1010 KP1005 KP1009 ec302 ec304 KP1004KP1008 EC1007 KP1007 KPX1001 pAmpC CTX-M-14 CTX-M-15 TEM-10 TEM-26 KPC-2KPC-2 KPC-3 OXA-10 OXA-2 (CMY-6) Clavulanic Acid 0.0548 0.247 0.0270.027 0.74 2.22 1.48 1.48 0.08 ND Tazobactam <0.0274 0.027 0.055 0.0270.74 2.22 0.74 4.44 0.0274 ND 3 X Z Z Z X Y X Z Z Z 4 X Y Z Z X X X Z ZZ 5 X Y Z Z X X X Z X Z 6 X X Z Z X X X Z Y Z 33 X Z Z Z Y Y X Z Y Z 34Z Z Z Z Y Y X Z Z Z 35 Z Z Z Z Z Z X Z Z Z 36 X X X X Y Y X Z Z Z 37 X XZ Z X X X Y Y X 38 X Y Z Y X X X Z Y Y 39 Y Y Z Y X Y X Z Y Z 40 X X Z YY Z Y Y X Y 41 Z Z Z Z X X X Z Z Z 42 Z Z Z Z X X X Z Z Z 43 Y Z Z Z X XX Z Y Z 45 Z Z Z Z Y Y X Z Z Z 46 Z Z Z Z X X X Z Z Z 47 Z Z Z Z Y Y X ZZ Z 48 Y Z Z Z X Y X Z Y Z 49 Y Z Z Z X X X Z Y Z 50 Y Z Z Z X X X Z Z Z51 Z Z Z Z X X X Z Y Y 52 Z Z Z Z X Y Y Z Z Z 53 Z Z Z Z X Y Y Z Y Z 54X Z Z Z X X X Z Y Y 55 Y Z Z Y X X X Z Y Z 56 X Y Z Z X X X Z Y Y 57 X ZZ Z X X X Z Z Z 58 Y Z Z Z X X X Z Z Z 59 Z Z Z Z Y X Y Z Y Z 60 Y Y Z ZX X X Z Y Y 61 Y Z Z Z X X X Z Y Z 62 Y Y Z Y Y X X Z Z Z 63 Y Y Z Z Y YY Z Y Y 65 Y Z Z Z Y Y Z Z Y Z 66 Y Z Z Z X X X Z X Z X = IC₅₀ of lessthan 0.5 μg/mL. Y = IC₅₀ of 0.5 μg/mL to 2 μg/mL. Z = IC₅₀ of greaterthan 2 μg/mL. ND = Not Determined.

Example 13

Selected β-lactamase inhibitors were also tested for their ability topotentiate the monobactam tigemonam. The potentiation effect is observedas the ability of BLI compounds to inhibit growth in the presence ofsub-inhibitory concentration of tigemonam. MIC of test strains vary from8 μg/mL to >128 μg/mL. Tigemonam is present in the test medium at 4μg/mL. Compounds tested at the highest concentration of 40 μg/mL. Inthis assay potency of compounds is determined as a concentration of BLIsto inhibit growth of bacteria in the presence of 4 μg/mL of aztreonam(MPC_(@4)). Tables 12 and 13 summarize BLI potency of tigemonampotentiation (MPC_(@4)) for various strains overexpressing class A(ESBLs), class C and class D beta-lactamases, respectively. TigemonamMIC for each strain is also shown. Table 12 summarizes activity of BLIsto potentiate tigemonam against strains expressing class A ESBLs. Table13 summarizes activity of BLIs to potentiate aztreonam against strainsexpressing class C and D enzymes.

TABLE 12 Tigemonam MIC (μg/mL) 512 256 >512 256 64 256 >512 512 MPC₄MPC₄ MPC₄ MPC₄ MPC₄ MPC₄ MPC₄ MPC₄ CTX-M-14 CTX-M-15 SHV-5 SHV-12 SHV-18TEM-10 TEM-10 TEM-26 KP1005 KP1009 ec308 KP1010 KP1012 EC1009 ec302ec304 Tazobactam Clavulanic 10 10 5 1.25 1.25 2.5 5 1.25 Acid 2.5 1.25<=0.6 <=0.6 <=0.6 <=0.6 2.5 <=0.6 5 Z Z Z Z Z Z Z Z 9 Z Z Z Z Z Z Z Z 18X X Y X X X Y Y 37 Z Z Z Z Z Z Z Z 48 Z Z Z Z Z Z Z Z 63 Z Y Z Y Y Z Z Z64 Z Y Y X Y Y Z Z 67 Z Y Z Y Y Z Z Z 68 Y Y Y X X Y Y Y X = MPC_(@4) ofless than 2 μg/mL. Y = MPC_(@4) of 2 μg/mL to 10 μg/mL. Z = MPC_(@4) ofgreater than 10 μg/mL. ND = Not Determined.

TABLE 13 Class C C C D S Tigemonam MIC (μg/mL) 16 256 8 32 MPC₄ 8 MPC₄MPC₄ MPC₄ CMY-6, MPC₄ OXA-10, OXA-2, ECL1002 EC1010 PAM2035 KP1007KPX1001 Tazobactam 10 2.5 5 5 40 Clavulanic >40 40 >40 <=0.6 1.25 Acid 5Y X X Z X 9 Y Y Y Z X 18 Y X X Y Y 37 X X X Z X 48 Y X Y Z X 63 Y X Y YX 64 X X Y X Y 67 Y X X Z X 68 Y X Y X X X = MPC_(@4) of less than 2μg/mL. Y = MPC_(@4) of 2 μg/mL to 10 μg/mL. Z = MPC_(@4) of greater than10 μg/mL. ND = Not Determined.

Example 14

Checkerboard assays were used to evaluate the ability of Compound 5 topotentiate various carbapenems (biapenem, doripenem, ertapenem,imipenem, and meropenem) against the strains expressing KPC alone or incombination with additional beta-lactamases. The highest concentrationof Compound 5 was 10 mg/L. The results are present in the Table 14.Compound 5 was capable to significantly potentiate multiple carbapenems.

TABLE 14 Concentration of Compound 5 (mg/L) to Potentiate Carbapenem(mg/L) Organism Strain Enzymes Antibiotic 0 0.16 0.31 0.625 1.25 2.5 510 Klebsiella KP1004 KPC-2 Biapenem Z X X X X X X X pneumoniaeKlebsiella KP1004 KPC-2 Doripenem Y Y X X X X X X pneumoniae KlebsiellaKP1004 KPC-2 Ertapenem Z Z Y Y X X X X pneumoniae Klebsiella KP1004KPC-2 Imipenem Z X X X X X X X pneumoniae Klebsiella KP1004 KPC-2Meropenem Z Y Y X X X X X pneumoniae Klebsiella KP1008 KPC-2 Biapenem ZX X X X X NG NG pneumoniae Klebsiella KP1008 KPC-2 Doripenem Y X X X X XNG NG pneumoniae Klebsiella KP1008 KPC-2 Ertapenem Z X X X X X NG NGpneumoniae Klebsiella KP1008 KPC-2 Imipenem Y X X X X X NG NG pneumoniaeKlebsiella KP1008 KPC-2 Meropenem Y X X X X X NG NG pneumoniaeKlebsiella KP1082 KPC-2, Biapenem Y X X X X X X X pneumoniae SHV-1Klebsiella KP1082 KPC-2, Doripenem Y X X X X X X X pneumoniae SHV-1Klebsiella KP1082 KPC-2, Ertapenem Y X X X X X X X pneumoniae SHV-1Klebsiella KP1082 KPC-2, Imipenem Y X X X X X X X pneumoniae SHV-1Klebsiella KP1082 KPC-2, Meropenem Y X X X X X X X pneumoniae SHV-1Klebsiella KP1087 KPC-2, Biapenem Z Z Z Z Y Y X X pneumoniae CTX-M-15,SHV-11, TEM-1 Klebsiella KP1087 KPC-2, Doripenem Z Z Z Z Z Y Y Xpneumoniae CTX-M-15, SHV-11, TEM-1 Klebsiella KP1087 KPC-2, Ertapenem ZZ Z Z Z Z Y Y pneumoniae CTX-M-15, SHV-11, TEM-1 Klebsiella KP1087KPC-2, Imipenem Z Y Y Y Y Y X X pneumoniae CTX-M-15, SHV-11, TEM-1Klebsiella KP1087 KPC-2, Meropenem Z Z Z Z Z Y Y X pneumoniae CTX-M-15,SHV-11, TEM-1 Klebsiella KX1019 KPC-2, Biapenem Z Y Y Y Y Y X X oxytocaOXA-2 Klebsiella KX1019 KPC-2, Doripenem Y Y Y Y X X X X oxytoca OXA-2Klebsiella KX1019 KPC-2, Ertapenem Z Y Y Y Y Y X X oxytoca OXA-2Klebsiella KX1019 KPC-2, Imipenem Y Y Y Y X X X X oxytoca OXA-2Klebsiella KX1019 KPC-2, Meropenem Y Y Y X X X X X oxytoca OXA-2Klebsiella KX1017 KPC-2, Biapenem Y Y Y X X X X X oxytoca OXA-2, SHV-30Klebsiella KX1017 KPC-2, Doripenem Y Y Y Y X X X X oxytoca OXA-2, SHV-30Klebsiella KX1017 KPC-2, Ertapenem Z Y Y Y Y X X X oxytoca OXA-2, SHV-30Klebsiella KX1017 KPC-2, Imipenem Z Y X X X X X X oxytoca OXA-2, SHV-30Klebsiella KX1017 KPC-2, Meropenem Y Y Y X X X X X oxytoca OXA-2, SHV-30Klebsiella KX1018 KPC-2, Biapenem Z X X X X X NG NG oxytoca SHV-40,OXY-1 Klebsiella KX1018 KPC-2, Doripenem Y X X X X X NG NG oxytocaSHV-40, OXY-1 Klebsiella KX1018 KPC-2, Ertapenem Z X X X X X NG NGoxytoca SHV-40, OXY-1 Klebsiella KX1018 KPC-2, Imipenem Y X X X X X NGNG oxytoca SHV-40, OXY-1 Klebsiella KX1018 KPC-2, Meropenem Y X X X X XNG NG oxytoca SHV-40, OXY-1 Escherichia EC1007 KPC-3 Biapenem Z X X X XX X X coli Escherichia EC1007 KPC-3 Doripenem Y X X X X X X X coliEscherichia EC1007 KPC-3 Ertapenem Z X X X X X X X coli EscherichiaEC1007 KPC-3 Imipenem Z X X X X X X X coli Escherichia EC1007 KPC-3Meropenem Y X X X X X X X coli Enterobacter ECL1058 KPC-3, Biapenem Z YY Y X X X X cloacae SHV-11, TEM-1 Enterobacter ECL1058 KPC-3, DoripenemZ Y Y Y Y X X X cloacae SHV-11, TEM-1 Enterobacter ECL1058 KPC-3,Ertapenem Z Z Z Z Y Y X X cloacae SHV-11, TEM-1 Enterobacter ECL1058KPC-3, Imipenem Z Y Y Y X X X X cloacae SHV-11, TEM-1 EnterobacterECL1058 KPC-3, Meropenem Z Y Y Y Y X X X cloacae SHV-11, TEM-1Enterobacter ECL1059 KPC-3, Biapenem Y X X X X X X X cloacae SHV-12,TEM-1 Enterobacter ECL1059 KPC-3, Doripenem Y X X X X X X X cloacaeSHV-12, TEM-1 Enterobacter ECL1059 KPC-3, Ertapenem Y X X X X X X Xcloacae SHV-12, TEM-1 Enterobacter ECL1059 KPC-3, Imipenem Y X X X X X XX cloacae SHV-12, TEM-1 Enterobacter ECL1059 KPC-3, Meropenem Y X X X XX X X cloacae SHV-12, TEM-1 Klebsiella KP1083 KPC-3, Biapenem Z Y X X XX X X pneumoniae SHV-1, TEM-1 Klebsiella KP1083 KPC-3, Doripenem Z Y X XX X X X pneumoniae SHV-1, TEM-1 Klebsiella KP1083 KPC-3, Ertapenem Z Y XX X X X X pneumoniae SHV-1, TEM-1 Klebsiella KP1083 KPC-3, Imipenem Z YX X X X X X pneumoniae SHV-1, TEM-1 Klebsiella KP1083 KPC-3, Meropenem ZY X X X X X X pneumoniae SHV-1, TEM-1 Klebsiella KP1084 KPC-3, BiapenemZ Z Z Z Z Y X X pneumoniae SHV-11, TEM-1 Klebsiella KP1084 KPC-3,Doripenem Z Z Z Z Y Y Y X pneumoniae SHV-11, TEM-1 Klebsiella KP1084KPC-3, Ertapenem Z Z Z Z Z Z Y Y pneumoniae SHV-11, TEM-1 KlebsiellaKP1084 KPC-3, Imipenem Z Z Z Y Y Y X X pneumoniae SHV-11, TEM-1Klebsiella KP1084 KPC-3, Meropenem Z Z Z Z Z Y Y X pneumoniae SHV-11,TEM-1 Klebsiella KP1088 KPC-3, Biapenem Z Y X X X X X X pneumoniaeSHV-11, TEM-1 Klebsiella KP1088 KPC-3, Doripenem Y Y Y X X X X Xpneumoniae SHV-11, TEM-1 Klebsiella KP1088 KPC-3, Ertapenem Z Z Y X X XX X pneumoniae SHV-11, TEM-1 Klebsiella KP1088 KPC-3, Imipenem Z Y X X XX X X pneumoniae SHV-11, TEM-1 Klebsiella KP1088 KPC-3, Meropenem Z Y YX X X X X pneumoniae SHV-11, TEM-l X = MIC of less than 0.5 mg/L. Y =MIC of 0.5 mg/L to 4 mg/L. Z = MIC of greater than 4 mg/L. NG = NoGrowth.

Example 15

An in vivo model can be used to evaluate the single dose pharmacokineticproperties and absolute oral bioavailability of a test compound. Asdescribed more specifically below, a test compound is administered toSprague-Dawley (SD) rats either intravenously or orally in a crossoverstudy design and the resulting pharmacokinetic properties and oralbioavailability are measured.

For intravenous administration, male rats were given a 30 minutesintravenous infusion dose of 20 or 50 mg/kg of Compound 5 via femoralvein cannula. Plasma samples (0.3 ml) were collected from jugular veincannula at 0.17, 0.33, 0.47, 0.58, 0.68, 0.75, 1, 2, 3, 4, and 6 hrsafter the dosing. For oral administration, male rats were given 50 mg/kgof Compound 5 (in saline) or Compound 62 (in 100% ethanol) orally usingan oral gavage tip. Plasma samples were collected from each rat at 0.08,0.17, 0.25, 0.33, 0.50, 0.75, 1, 2, 3, 4, and 6 hrs after the dosing.

Plasma concentrations of the compounds were tested using LC/MS/MS methodwith a lower limit of quantification of 10 ng/mL for Compound 5 and 100ng/mL for Compound 62. Extraction: 50 μL volumes of plasma from samplesand standards were extracted using 200 μL of methanol with 100 mMammonium acetate, 2 g/mL gatifloxacin (internal standard for Compound62) and 2 ug/mL Compound 38 (internal standard for Compound 5). Thesamples were mixed and centrifuged for 30 min at 3000×g. 150 μL ofsupernatant was removed and added to 450 μL of water.

HPLC—mass spectrometry: An Agilent 1100HPLC pump, HTC PAL autosamplerand a Sciex 3200Q mass spectrometer were used for separation andquantification. Compound 62 and its internal standard were detectedusing+ESI. Compound 5 and its internal standard were detected using—ESI.LC/MS/MS: 1) Column: Chromolith FastGradient RP-18e, 50×2 mm; 2) Mobilephase A: Aqueous Water with 0.1% TFA, Orgainic phase B: Acetonitrilewith 0.1% TFA; Flow Rate: 600 μL/min; Injection volume: 10 μL; HPLCgradient: 5% B→60% B, 0.01→1.5 min; 60% B, 1.5→1.6 min; 60% B→5% B,1.6→1.7 min; 5% B, 1.7→2.7 min.

Plasma concentrations were modeled using WinNonlin® (Pharsight Corp,Mountain View, Calif.).

In this experiment, three male Sprague Dawley rats were given Compound 5by intravenous or oral route. At designated time points, bloods werecollected and analyzed. As shown in the above Table 15 and FIG. 1,Compound 5 has a linear PK in rats. However, Compound 5 it is not orallyabsorbed.

TABLE 15 Cmax CL/F AUC Route of Adm Dose/(mg/kg) T_(1/2) (hr) (mg/L)(L/h/kg) (mg * h/L) IV 20 1.56 19.82 1.65 12.15 IV 50 4.53 45.93 1.7728.19 PO 50 1.55 0.29 60.38 0.81

In this experiment, three male Sprague dawley rats were given Compound 5by intravenous or Compound 62 orally (pro-drug for Compound 5). Plasmasamples were collected at designated time points and analyzed for thepresence of Compound 5. This study was designed to determine the oralbioavailability of Compound 62 a pro-drug of Compound 5. Male rats(non-fasted) were orally administered 50 mg/kg of the prodrug Compound62. As shown in FIG. 2, the pro-drug of Compound 5 has oralbioavailability of greater than 80%.

Polymorphs can be detected, identified, classified and characterizedusing well-known techniques such as, but not limited to, differentialscanning calorimetry (DSC), thermogravimetry (TGA) and powder X-raydiffractometry (PXRD).

Example 16

The crystal structure of Compound 5 was analyzed using X-ray powderdiffraction (“PXRD”). The X-ray diffraction data were collected at roomtemperature using a PANalytical X'Pert Pro diffractometer (Cu Kαradiation) fitted with an automatic sample changer, a theta-thetagoniometer, automatic beam divergence slits, a secondary monochromatorand a scintillation counter. Samples were prepared for analysis bypacking the powder into a 12 mm diameter, 0.25 mm deep cavity that hadbeen cut into a Si zero-background wafer specimen mount. The sample wasrotated while being irradiated with copper K-alpha 1 X-rays(wavelength=1.5406 Ångstroms) with the X-ray tube operated at 45 kV/40mA. The analyses were performed with the goniometer running incontinuous mode set for a 5 second count per 0.02° step over a two thetarange of 2° to 550. The illustrative PXRD pattern for Compound 5 isshown in FIG. 3.

As will be appreciated by the skilled crystallographer, the relativeintensities of the various peaks reported in FIG. 3 may vary due to anumber of factors such as orientation effects of crystals in the X-raybeam or the purity of the material being analyzed or the degree ofcrystallinity of the sample. The peak positions may also shift forvariations in sample height but the peak positions will remainsubstantially as defined in FIG. 3. The skilled crystallographer alsowill appreciate that measurements using a different wavelength willresult in different shifts according to the Bragg equation—nλ=2d sin θ.Such further PXRD patterns generated by use of alternative wavelengthsare considered to be alternative representations of the PXRD patterns ofthe crystalline materials of the present invention and as such arewithin the scope of the present invention.

Table 16 lists peak positions and relative intensities for the PXRDpattern of FIG. 3. Accordingly, some embodiments include a crystallineform of Compound 5 having three or more, four or more, five or more, sixor more, seven or more, eight or more, nine or more, or ten or morecharacteristic PXRD (wavelength=1.5406 Å) peaks selected from 9.0°,15.7°, 17.3°, 17.6°, 18.1°, 21.3°, 22.4°, 23.5°, 24.9°, 27.2°, 27.4°,28.1°, 29.1°, 31.2°, and 35.7° 20. Some embodiments include acrystalline form of Compound 5 having three or more, four or more, fiveor more, or six characteristic PXRD (wavelength=1.5406 Å) peaks selectedfrom 9.0°, 17.3°, 17.6°, 18.1°, 22.4°, and 27.2° 20. Some embodimentsinclude a crystalline form of Compound 5 having characteristic PXRD(wavelength=1.5406 Å) peaks at 9.1°, 17.3°, 17.6°, and 18.1° 20.

TABLE 16 °2θ Area [cts °2θ] d-spacing [Å] 9.0088 870.8 9.80831 12.013229.19 7.36118 13.2369 19.12 6.68332 15.4527 55.73 5.72961 16.6911 41.975.30719 17.3464 285.76 5.10815 17.59 171.25 5.03794 18.1212 475.594.89145 19.9585 23.95 4.4451 20.1214 18.12 4.40949 21.3328 84.5 4.1617522.4035 147.38 3.96521 22.9212 39.45 3.87681 23.48 60.99 3.78579 24.888177.52 3.5747 26.1352 20.92 3.40689 26.3458 20.23 3.38013 27.2278 162.93.27261 27.357 50.29 3.25744 28.0871 54.62 3.17441 29.0644 51.29 3.0698529.63 30.23 3.01253 30.1989 19.34 2.95706 31.2457 65.66 2.86033 32.164132.04 2.78073 33.7983 19.84 2.64992 35.1614 21.23 2.55025 35.6871 57.82.51388 36.5979 22 2.45338 37.7599 33.73 2.3805 39.8439 31.99 2.26066

As is well understood in the art, because of the experimentalvariability when X-ray diffraction patterns are measured on differentinstruments, the peak positions are assumed to be equal if the two theta(20) values agree to within 0.2° (i.e., +0.2°). For example, the UnitedStates Pharmacopeia states that if the angular setting of the 10strongest diffraction peaks agree to within ±0.2° with that of areference material, and the relative intensities of the peaks do notvary by more than 20%, the identity is confirmed. Accordingly, peakpositions within 0.2° of the positions recited herein are assumed to beidentical.

Example 17

DSC measure thermal transition temperatures at which a crystalline formabsorbs or releases heat when its crystal structure changes or it melts.TGA is used to measure thermal stability and the fraction of volatilecomponents of a sample by monitoring the weight change as the sample isheated. If infrared spectroscopy is conducted on the volatile componentsoutgassed during TGA analysis of a pseudopolymorph (TGA-IR), then themolecular composition of the pseudopolymorph can be determined. Thesetechniques are thus useful for characterizing solid state forms existingas solvates and/or hydrates.

Compound 5 was analyzed using differential scanning calorimetry (DSC). ATA Instruments Q100 differential scanning calorimeter equipped with anautosampler and a refrigerated cooling system under 40 mL/min N₂ purgewas used to perform the analysis. Each sample was heated from 25 to 300°C. at 15° C. per minute in an aluminium pan with the lid laid on top,with a nitrogen purge gas. The data from DSC analyses are dependent onseveral factors, including the rate of heating, the purity of thesample, crystal size, and sample size. The DSC thermogram obtained forthe sample of Compound 5 is shown in FIG. 4 overlayed with the TGAthermogram. These data reveal a single endothermic transition at 155° C.

Thermogravimetric-infrared (TG-IR) Analysis was performed on a TAInstruments Q5000 thermogravimetric analyzer interfaced to a Nicolet6700 FT-IR spectrometer (Thermo Electron) equipped with an externalTGA-IR module with a gas flow cell and DTGS detector. The FT-IRwavelength verification was performed using polystyrene, and the TGcalibration standards were nickel and Alumel™. The sample was placed ina platinum or aluminium sample pan, and the pan was inserted into the TGfurnace. The TG instrument was started first, immediately followed bythe FT-IR instrument. The TG instrument was operated under a flow ofhelium at 90 and 10 cc/min for the purge and balance, respectively. Thefurnace was heated under nitrogen at a rate of 15° C./minute to a finaltemperature of 230° C. IR spectra were collected approximately every 32seconds for approximately 13 minutes. Each IR spectrum used 32 co-addedscans collected at a spectral resolution of 4 cm⁻¹. The TGA thermogramobtained for the sample of Compound 5 is shown in FIG. 4 overlayed withthe DSC thermogram. These TGA data with IR analysis of the evolved gasindicate that the input material is non-solvated but loses onemole-equivalent of water between 135 and 181° C.

All references cited herein, including but not limited to published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

The above description discloses several methods and materials that aresusceptible to modifications, as well as alterations in the fabricationmethods and equipment. Such modifications will become apparent to thoseskilled in the art from a consideration of this disclosure or practiceof the methods disclosed herein. Consequently, it is not intended thatthis invention be limited to the specific embodiments disclosed herein,but that it cover all modifications and alternatives coming within thetrue scope and spirit of the invention.

What is claimed is:
 1. A sterile container comprising: an antibacterialagent in solid form; and a compound of Formula (I) in dolid form havingthe structure:

or a pharmaceutically acceptable salt thereof, wherein: Y is a 1-4 atomalkylene or 2-4 atom alkenylene linker, optionally substituted by one ormore substituents selected from the group consisting of Cl, F, CN, CF₃,—R⁹, —OR⁹, —C(═O)NR⁹R¹⁰, and —C(═O)OR⁹, wherein said alkylene oralkenylene linker is optionally fused to an optionally substituted aryl,optionally substituted heteroaryl, optionally substituted carbocyclyl,or optionally substituted heterocyclyl; R¹ is selected from a groupconsisting of —C₁₋₉alkyl, —C₂₋₉alkenyl, —C₂₋₉alkynyl, —NR⁹R¹⁰,—C₁₋₉alkylR¹¹, —C₂₋₉alkenylR¹¹, —C₂₋₉alkynylR¹¹, -carbocyclyl-R¹¹,—CH(OH)C₁₋₉alkylR⁹, —CH(OH)C₂₋₉alkenylR⁹, —CH(OH)C₂₋₉alkynylR⁹,—CH(OH)carbocyclyl-R⁹, —C(═O)R⁹, —C(═O)C₁₋₉alkylR⁹, —C(═O)C₂₋₉alkenylR⁹,—C(═O)C₂₋₉alkynylR⁹, —C(═O)C₂₋₉carbocyclyl-R⁹, —C(═O)NR⁹R¹⁰,—N(R⁹)C(═O)R⁹, —N(R⁹)C(═O)NR⁹R¹⁰, —N(R⁹)C(═O)OR⁹, —N(R⁹)C(═O)C(═NR¹⁰)R⁹,—N(R⁹)C(═O)C(═CR⁹R¹⁰)R⁹, —N(R⁹)C(═O)C₁₋₄alkylN(R⁹)C(═O)R⁹,—N(R⁹)C(═NR¹⁰)R⁹, —C(═NR¹⁰)NR⁹R¹⁰, —N═C(R⁹)NR⁹R¹⁰, —N(R⁹)SO₂R⁹,—N(R⁹)SO₂NR⁹R¹⁰, —N═CHR⁹, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted carbocyclyl, andoptionally substituted heterocyclyl; R⁶ is selected from a groupconsisting of H, —C₁₋₉alkyl, C₂₋₉alkenyl, —C₂₋₉alkynyl, carbocyclyl,—C₁₋₉alkylR¹¹, —C₂₋₉alkenylR¹¹, —C₂₋₉alkynylR¹¹, carbocyclyl-R¹¹,—C(═O)OR⁹, —C₁₋₉alkylCO₂R⁹, —C₂₋₉alkenylCO₂R⁹, —C₂₋₉alkynylCO₂R⁹, and-carbocyclyl-CO₂R⁹, or alternatively: (i) R⁶ and an R⁷ are takentogether with the atoms to which they are attached to form an optionallysubstituted carbocyclyl or optionally substituted heterocyclyl, or (ii)R⁶ is absent when the carbon to which it is attached is a ring atom inan aryl or heteroaryl ring; each R⁷ is independently selected from agroup consisting of H, halo, —C₁₋₉alkyl, —C₂₋₉alkenyl, —C₂₋₉alkynyl,—NR⁹R¹⁰, —OR⁹, —C₁₋₉alkylCO₂R⁹, —C₂₋₉alkenylCO₂R⁹, —C₂₋₉alkynylCO₂R⁹,and -carbocyclyl-CO₂R⁹, or independently: (i) R⁶ and an R⁷ are takentogether with the atoms to which they are attached to form an optionallysubstituted carbocyclyl or optionally substituted heterocyclyl, or (ii)R⁷ and an R⁸ are taken together with the atoms to which they areattached to form an optionally substituted carbocyclyl or optionallysubstituted heterocyclyl; each R⁸ is independently selected from a groupconsisting of H, halo, —C₁₋₉alkyl, —C₂₋₉alkenyl, —C₂₋₉alkynyl, —NR⁹R¹⁰,—OR⁹, —C₁₋₉alkylCO₂R⁹, —C₂₋₉alkenylCO₂R⁹, —C₂₋₉alkynylCO₂R⁹,-carbocyclyl-CO₂R⁹, or independently: (i) an R⁷ and an R⁸ are takentogether with the atoms to which they are attached to form an optionallysubstituted carbocyclyl or optionally substituted heterocyclyl, (ii) ageminal R⁷ and R⁸ together form —C₂₋₉alkenylenylCO₂R⁹, or (iii) each R⁸attached to a ring atom forming part of an optionally substituted arylis absent; each R⁹ is independently selected from a group consisting ofH, —C₁₋₉alkyl, C₂₋₉alkenyl, —C₂₋₉alkynyl, carbocyclyl, —C₁₋₉alkylR¹¹,—C₂₋₉alkenylR¹¹, —C₂₋₉alkynylR¹¹, -carbocyclyl-R¹¹, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted carbocyclyl, and optionally substituted heterocyclyl; eachR¹⁰ is independently selected from a group consisting of H, —C₁₋₉alkyl,—OR⁹, —CH(═NH), —C(═O)OR⁹, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted carbocyclyl, andoptionally substituted heterocyclyl; each R¹¹ is independently selectedfrom a group consisting of optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted carbocyclyl, andoptionally substituted heterocyclyl; X is selected from a groupconsisting of —CO₂R¹², and a carboxylic acid isostere; R¹² is selectedfrom a group consisting of H, C₁₋₉alkyl, —(CH₂)₀₋₃—R₁₁,—C(R¹³)₂OC(O)C₁₋₉alkyl, —C(R¹³)₂OC(O)R¹¹, —C(R¹³)₂OC(O)OC₁₋₉alkyl and—C(R¹³)₂OC(O)OR¹¹; each R¹³ is independently selected from a groupconsisting of H and C₁₋₄alkyl; and m is independently zero or an integerfrom 1 to 2, wherein each C₁₋₉alkyl, C₂₋₉alkenyl, and C₂₋₉alkynyl isindependently optionally substituted.
 2. The sterile container of claim1, wherein the compound has the structure of formula II:

or a pharmaceutically acceptable salt thereof, wherein: the bondrepresented by a dashed and solid line represents a bond selected fromthe group consisting of a single bond and a double bond with the provisothat the dashed and solid line can only be a double bond when n is 1; R¹is selected from a group consisting of —NR⁹R¹⁰, —C₁₋₉alkylR¹¹,—C₂₋₉alkenylR¹¹, —C₂₋₉alkynylR¹¹, -carbocyclyl-R¹¹, —CH(OH)C₁₋₉alkylR⁹,—CH(OH)C₂₋₉alkenylR⁹, —CH(OH)C₂₋₉alkynylR⁹, —CH(OH)carbocyclyl-R⁹,—C(═O)R⁹, —C(═O)C₁₋₉alkylR⁹, —C(═O)C₂₋₉alkenylR⁹, —C(═O)C₂₋₉alkynylR⁹,—C(═O)C₂₋₉carbocyclyl-R⁹, —C(═O)NR⁹R¹⁰, —N(R⁹)C(═O)R⁹,—N(R⁹)C(═O)NR⁹R¹⁰, —N(R⁹)C(═O)OR⁹, —N(R⁹)C(═O)C(═NR⁰)R⁹,—N(R⁹)C(═O)C(═CR⁹R¹⁰)R⁹, —N(R⁹)C(═O)C₁₋₄alkylN(R⁹)C(═O)R⁹,—N(R⁹)C(═NR¹⁰)R⁹, —C(═NR¹⁰)NR⁹R¹⁰, —N═C(R⁹)NR⁹R¹⁰, —N(R⁹)SO₂R⁹,—N(R⁹)SO₂NR⁹R¹⁰, —N═CHR⁹, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted carbocyclyl, andoptionally substituted heterocyclyl; R² and R⁴ are independentlyselected from a group consisting of H, Cl, F, CN, CF₃, —R⁹, —OR⁹,—C(═O)NR⁹R¹⁰, and —C(═O)OR⁹; R³ and R⁵ are independently selected from agroup consisting of H, Cl, F, CN, CF₃, —R⁹, —OR⁹, —C(═O)NR⁹R¹⁰, and—C(═O)OR⁹, with the proviso that if the bond represented by a dashed andsolid line is a double bond then R³ and R⁵ are absent; R⁶ is selectedfrom a group consisting of H, —C₁₋₉alkyl, C₂₋₉alkenyl, —C₂₋₉alkynyl,—C₁₋₉alkylR¹¹, —C₂₋₉alkenylR¹¹, —C₂₋₉alkynylR¹¹, —C(═O)OR⁹, and—C₁₋₉alkylCO₂R⁹, —C₂₋₉alkenylCO₂R⁹, and —C₂₋₉alkynylCO₂R⁹; each R⁷ isindependently selected from a group consisting of H, —NR⁹R¹⁰, —OR⁹, and—C₁₋₉alkylCO₂R⁹, —C₂₋₉alkenylCO₂R⁹, and —C₂₋₉alkynylCO₂R⁹; each R⁸ isindependently selected from a group consisting of H, —NR⁹R¹⁰, —OR⁹, and—C₁₋₉alkylCO₂R⁹, —C₂₋₉alkenylCO₂R⁹, and —C₂₋₉alkynylCO₂R⁹; each R⁹ isindependently selected from a group consisting of H, —C₁₋₉alkyl,C₂₋₉alkenyl, —C₂₋₉alkynyl, —C₁₋₉alkylR¹¹, —C₂₋₉alkenylR¹¹,—C₂₋₉alkynylR¹¹, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted —(CH₂)₀₋₃carbocyclyl, and optionallysubstituted heterocyclyl; each R¹⁰ is independently selected from agroup consisting of H, —C₁₋₉alkyl, —OR⁹, —CH(═NH), optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted carbocyclyl, and optionally substituted heterocyclyl; X is—CO₂H; and n is independently zero or an integer from 1 to
 2. 3. Thesterile container of claim 2, wherein n is
 1. 4. The sterile containerof claim 2, wherein R², R³, R⁴, and R⁵ are hydrogen.
 5. The sterilecontainer of claim 1, wherein the compound has the structure of formulaIIIa, IIIb, IVa, IVb, or IVc:

or a pharmaceutically acceptable salt thereof, wherein: the bondrepresented by a dashed and solid line represents a bond selected fromthe group consisting of a single bond and a double bond; each R² and R⁴are independently selected from a group consisting of H, Cl, F, CN, CF₃,—R⁹, —OR⁹, —C(═O)NR⁹R¹⁰, and —C(═O)OR⁹; and each R³ and R⁵ areindependently selected from a group consisting of H, Cl, F, CN, CF₃,—R⁹, —OR⁹, —C(═O)NR⁹R¹⁰, and —C(═O)OR⁹, with the proviso that if thebond represented by a dashed and solid line is a double bond then R³ andR⁵ are absent.
 6. The sterile container of claim 1, wherein R¹ is—NHC(═O)C₁₋₉alkylR¹¹.
 7. The sterile container of claim 6, wherein R¹¹is optionally substituted aryl or optionally substituted heteroaryl. 8.The sterile container of claim 7, wherein R¹¹ is thien-2-yl.
 9. Thesterile container of claim 1, wherein m is
 1. 10. The sterile containerof claim 1, wherein R⁶ and each R⁷ and R⁸ is hydrogen.
 11. The sterilecontainer of claim 1, wherein X is —CO₂H.
 12. The sterile container ofclaim 1, wherein the compounds has the structure selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof.
 13. The sterile containerof claim 1, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 14. The sterile containerof claim 1, wherein the antibacterial agent is a β-lactam antibacterialagent.
 15. The sterile container of claim 14, wherein the β-lactamantibacterial agent is a carbapenem selected from the group consistingof simipenem, biapenem, doripenem, meropenem, and ertapenem.
 16. Thesterile container of claim 15, wherein the carbapenem is meropenem. 17.The sterile container of claim 14, wherein the β-lactam antibacterialagent is selected from the group consisting of Amoxicillin, Ampicillin,Pivampicillin, Hetacillin, Bacampicillin, Metampicillin, Talampicillin,Epicillin, Carbenicillin, Carindacillin, Ticarcillin, Temocillin,Azlocillin, Piperacillin, Mezlocillin, Mecillinam, Pivmecillinam,Sulbenicillin, Benzylpenicillin (G), Clometocillin, Benzathinebenzylpenicillin, Procaine benzylpenicillin, Azidocillin, Penamecillin,Phenoxymethylpenicillin (V), Propicillin, Benzathinephenoxymethylpenicillin, Pheneticillin, Cloxacillin, Dicloxacillin,Flucloxacillin, Oxacillin, Meticillin, Nafcillin, Faropenem, Biapenem,Doripenem, Ertapenem, Imipenem, Meropenem, Panipenem, Tomopenem,Razupenem, Cefazolin, Cefacetrile, Cefadroxil, Cefalexin, Cefaloglycin,Cefalonium, Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, Cefazedone,Cefazaflur, Cefradine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole,Cefminox, Cefonicid, Ceforanide, Cefotiam, Cefprozil, Cefbuperazone,Cefuroxime, Cefuzonam, Cefoxitin, Cefotetan, Cefmetazole, Loracarbef,Cefixime, Ceftazidime, Ceftriaxone, Cefcapene, Cefdaloxime, Cefdinir,Cefditoren, Cefetamet, Cefmenoxime, Cefodizime, Cefoperazone,Cefotaxime, Cefpimizole, Cefpiramide, Cefpodoxime, Cefsulodin, Cefteram,Ceftibuten, Cefatiolene, Ceftizoxime, Flomoxef, Latamoxef, Cefepime,Cefozopran, Cefpirome, Cefquinome, Ceftobiprole, Ceftaroline, CXA-101,RWJ-54428, RWJ-333441, ME1036, BAL30072, BAL19764, Ceftiofur,Cefquinome, Cefovecin, Aztreonam, Tigemonam, Carumonam, RWJ-442831,RWJ-333441, and RWJ-333442.
 18. The sterile container of claim 1,wherein the compound and the antibacterial agent are blended.
 19. Thesterile container of claim 1, wherein the compound and the antibacterialagent are not blended.
 20. The sterile container of claim 1, wherein thecompound is in crystalline form.
 21. The sterile container of claim 1,wherein the antibacterial agent is in crystalline form.
 22. The sterilecontainer of claim 1, wherein the compound and the antimicrobial agentare lyophiles.
 23. The sterile container of claim 1, wherein the molarratio of compound to antibacterial agent is from 1:8 to 8:1.
 24. Thesterile container of claim 1, wherein the molar ratio of compound toantibacterial agent is from 1:2 to 2:1.
 25. The sterile container ofclaim 1, wherein the molar ratio of compound to antibacterial agent is1:1.
 26. The sterile container of claim 1, further comprising a pHadjuster.
 27. The sterile container of claim 26, wherein the pH adjusteris selected from the group consisting of NaOH, citric acid, and sodiumcarbonate.
 28. The sterile container of claim 1, wherein theantibacterial agent comprises meropenem, and the compound is:

or a pharmaceutically acceptable salt thereof.
 29. A sterile containercomprising: meropenem and a compound having a structure of:

or a pharmaceutically acceptable salt thereof.
 30. The sterile containerof claim 29, further comprising saline and sodium carbonate.
 31. Amethod of preparing a pharmaceutical composition for administration,comprising reconstituting the contents of the sterile container of claim1 using a pharmaceutically acceptable diluent.
 32. The method of claim31, wherein the diluent is selected from the group consisting of asaline solution, a dextrose solution, and a sodium carbonate solution.33. The method of claim 31, wherein the antibacterial agent comprisesmeropenem, and the compound is:

or a pharmaceutically acceptable salt thereof.
 34. A method ofadministration comprising administering the reconstituted solution ofclaim 31 intravenously to a patient.
 35. The method of claim 34, whereinthe antibacterial agent comprises meropenem, and the compound is:

or a pharmaceutically acceptable salt thereof.